Antibodies comprising chimeric constant domains

ABSTRACT

Antibodies, antigen-binding proteins and Fc-fusion proteins that comprise recombinant polypeptides containing a chimeric heavy chain constant region sequence are provided that bind to certain Fc receptors however have reduced effector functions. Methods of making constructs for expression of such chimeric Fc-containing antibodies, antigen-binding proteins and Fc-fusion proteins in cell systems, and methods of producing and isolating the chimeric Fc-containing proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/759,578, filed 1 Feb. 2013, whichapplication is herein incorporated by reference.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 8550US_ST25.txt created onJan. 29, 2014 (42,443 bytes).

FIELD OF THE INVENTION

The present invention concerns antibodies or antigen-binding proteinsengineered with recombinant polypeptides comprising a chimeric constantregion, more specifically including a chimeric hinge region in the heavychain constant region. The present invention relates to antibodies andantigen-binding proteins comprising such recombinant polypeptides thatreduce effector functions and provide an advantage for use in therapy.

BACKGROUND OF THE INVENTION

Immunoglobulins of the IgG class are attractive as therapeutic agents.IgGs exists as four subclasses in humans, IgG1, IgG2, IgG3, and IgG4.The heavy chain constant (C_(H)) region of IgG comprises three domains,C_(H)1, C_(H)2, C_(H)3, where C_(H)1 and C_(H)2 are linked by a hinge.Although the role of each subclass appears to vary between species, itis known that the heavy chain constant domain is responsible for variousbiological effector functions. The human IgG subclasses mediate aplethora of cellular immune responses through their interaction with Fcγreceptors (FcγRs), such as cell killing, complement activation,phagocytosis and opsonization. In an attempt to understand andmanipulate the effects of IgG subclass binding to FcγRs, researchershave made various mutations to the constant domains of IgGs and studiedthe resulting IgG/FcγR interaction (see e.g. Canfield and Morrison J ExpMed 73, 1483-1491 (1991); Chappel, M. S., et al. JBC 268(33), 25124-31(1993); and Armour, K. L., et al. Eur J Immunol 29, 2613-24 (1999)).

Fc dependent cytotoxic activity of human IgG antibodies requires bindingof the Fc region of the antibody (which consists of at least afunctional CH2 and CH3 domain) to an FcγR on the surface of an effectorcell, such as a natural killer cell, an activated macrophage or thelike. Complement-mediated lysis can also be triggered by the interactionof the Fc region with various complement components. With regard to FcγRbinding, it has been suggested that several amino acid residues in thehinge region and in the C_(H)2 domain of the antibody are important (seeSarmay, G, et al. Mol Immunol 29, 633-9 (1992); Greenwood, J et al.,Eur. J. Immunol, 23(5), 1098 (1993), Morgan, A. et al, Immunology, 86,319 (1995), Stevenson, G T, Chemical Immunology, 65, 57-72 (1997)).Glycosylation of a site (N297) in the CH2 domain and variations in thecomposition of its carbohydrates also strongly affect the IgG/FcγRinteraction (Stevenson, G T, Chemical Immunology, 65, 57-72 (1997);Sibéril et al Immunol Ltrs 106, 111-118 (2006)).

For certain antibody therapies, it may be advantageous to engineer theFc receptor binding properties so as to activate all, some, or none ofthe available effector mechanisms, without affecting the antibody'spharmacokinetic properties. The desired combination of therapeuticproperties may not be available in the natural antibody repertoire. Thedesign of antibodies with reduced effector function should beefficacious for example when the therapeutic objective is not to kill atarget cell, but to block or activate a cell surface molecule on itssurface without triggering cytotoxicity. Another setting in whichreduced binding to Fc receptors could be desirable is when the antibodyis bispecific, and its desired therapeutic properties arise from thedifferent binding specificities. For example, a common usage ofbispecific antibodies is to combine a tumor targeting specificity with aT cell activating specificity in order to trigger tumor-specific T cellkilling. In this case, if the Fc portion binds to an Fc receptor, thenpotentially that could trigger undesirable killing of cells bearing Fcreceptors by T cells, or killing of T cells by Fc receptor-bearing cellssuch as natural killer cells or macrophages.

Thus, there exists a need for improved biological therapies, such asantibodies with desirable properties for use in such therapies.Applicants have discovered that recombinant proteins containingsubstituted or otherwise modified antibody heavy chains, includingrecombinant antibodies and recombinant receptor-Fc fusion proteins, havealtered Fc receptor binding activity, which reduce the risk of unwantedside effects, and thus provide improved therapeutic effect.

SUMMARY OF THE INVENTION

The antibodies, antigen-binding proteins and Fc-fusion proteins that aredisclosed herein are engineered to have reduced binding to Fc receptors.

One aspect of the invention provides a recombinant polypeptidecomprising a chimeric Fc region, wherein the Fc region comprises achimeric hinge comprising the amino acid sequence EPKSCDKTHTCPPCPAPPVA(SEQ ID NO: 8). The invention also provides a recombinant polypeptidecomprising a chimeric Fc region, wherein the Fc region comprises achimeric hinge comprising the amino acid sequence ESKYGPPCPPCPAPPVA (SEQID NO: 9).

Another aspect of the invention provides a recombinant polypeptidecomprising a chimeric Fc region, wherein the Fc region comprises achimeric hinge comprising the amino acid sequence EPKSCDKTHTCPPCPAPPVA(SEQ ID NO: 8) linked to an IgG4 CH2 region. Still another aspect of theinvention provides a recombinant polypeptide comprising a chimeric Fcregion, wherein the Fc region comprises a chimeric hinge comprising theamino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9) linked to an IgG4CH2 region.

In some embodiments, the recombinant polypeptide comprises a chimeric Fcregion, wherein the Fc region comprises an IgG1 or IgG4 CH3 region, or avariant thereof. In other embodiments, the recombinant polypeptidecomprises a chimeric Fc region, wherein the Fc region binds to FcγRIIA.In other aspects the recombinant polypeptide comprises a chimeric Fcregion, wherein the Fc region binds to FcγRIIA and FcγRIIB.

In other embodiments, the invention provides a recombinant polypeptidecomprising a chimeric Fc region, wherein the Fc region compriseschimeric hinge comprising an amino acid sequence EPKSCDKTHTCPPCPAPPVA(SEQ ID NO: 8) and the recombinant polypeptide binds to FcγRIIA.

In still other aspects, the invention provides a recombinant polypeptidecomprising a chimeric Fc region, wherein the Fc region comprises achimeric hinge comprising an amino acid sequence ESKYGPPCPPCPAPPVA (SEQID NO: 9) and the recombinant polypeptide binds to FcγRIIA.

A further aspect of the invention provides a recombinant polypeptidecomprising a heavy chain constant (CH) region comprising, fromN-terminus to C-terminus, a CH1 domain, a chimeric hinge, a CH2 domain,and a CH3 domain wherein the CH1 domain comprises a human IgG1 CH1 or ahuman IgG4 CH1 having at least the amino acid sequence DKKV or DKRV frompositions 212 to 215 (EU numbering), the chimeric hinge comprises ahuman IgG1 or a human IgG4 upper hinge amino acid sequence frompositions 216 to 227 (EU numbering) and a human IgG2 lower hinge aminoacid sequence PCPAPPVA (SEQ ID NO: 3) from positions 228 to 236 (EUnumbering), the CH2 domain comprises a human IgG4 CH2 domain amino acidsequence from positions 237 to 340 (EU numbering), and the CH3 domaincomprises a human IgG1 or a human IgG4 CH3 domain sequence frompositions 341 to 447 (EU numbering), or a variant thereof.

Some embodiments of the invention provide a recombinant polypeptidewherein the CH1 domain comprises a human IgG1 CH1 amino acid sequence(SEQ ID NO: 43), and the chimeric hinge comprises the amino acidsequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8). Still another embodimentof the invention provides a recombinant polypeptide wherein the CH1domain comprises a human IgG4 CH1 amino acid sequence (SEQ ID NO: 44),and the chimeric hinge comprises the amino acid sequenceESKYGPPCPPCPAPPVA (SEQ ID NO: 9).

Another embodiment of the invention provides a recombinant polypeptidewherein the CH1 domain comprises the amino acid sequence DKKV (SEQ IDNO: 4), and the chimeric hinge comprises the amino acid sequenceEPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8). Still another embodiment of theinvention provides a recombinant polypeptide wherein the CH1 domaincomprises the amino acid sequence DKRV (SEQ ID NO: 5), and the chimerichinge comprises the amino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO:9). In another aspect, the CH1 domain comprises a variant of SEQ ID NO:43 or 44.

Another embodiment of the invention provides a recombinant polypeptidewherein the CH2 domain comprises the amino acid sequence SEQ ID NO: 10.Yet another embodiment of the invention provides a recombinantpolypeptide wherein the CH3 domain comprises the amino acid sequence SEQID NO: 11 or SEQ ID NO: 12. In another aspect, the CH3 domain comprisesSEQ ID NO: 41 or 42.

An aspect of the invention provides a recombinant polypeptide, whereinthe polypeptide comprises N′-VD1-X1_(n)-Y1-Y2-X2-X3-C′, wherein:

N′ is the N-terminus and C′ is the C-terminus of the polypeptide,VD1 is an amino acid sequence comprising an antigen-binding domain,X1 is an amino acid sequence comprising a domain selected from the groupconsisting of an IgG1 CH1 domain or a variant thereof, an IgG4 CH1domain or a variant thereof, and at least positions 212-215 (EUnumbering) of an IgG1 or IgG4 CH1 domain,Y1 comprises an amino acid sequence from positions 216-227 (EUnumbering) of an IgG1 or IgG4 hinge region,Y2 comprises the human IgG2 lower hinge region amino acid sequencePCPAPPVA (SEQ ID NO: 3) from positions 228 to 236 (EU numbering),X2 is an amino acid sequence comprising an IgG4 CH2 domain, or a variantthereof, andX3 is an amino acid sequence comprising an IgG1 CH3 domain or an IgG4CH3 domain, or a variant thereof; wherein n=0 or 1.

In some embodiments of the invention, n=1. In yet another embodiment ofthe invention, X1 comprises the amino acid sequence DKKV (SEQ ID NO: 4),and Y1-Y2 comprises a chimeric hinge consisting of the amino acidsequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8). In still anotherembodiment of the invention, X1 comprises the amino acid sequence DKRV(SEQ ID NO: 5), and Y1-Y2 comprises a chimeric hinge consisting of theamino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9). In one moreembodiment, X1 comprises SEQ ID NO 43 or SEQ ID NO: 44. In anotheraspect of the invention, X2 comprises SEQ ID NO: 10. In anotherembodiment of the invention, X3 comprises SEQ ID NO: 11 or SEQ ID NO:12. In yet another embodiment, X3 comprises SEQ ID NO: 41 or SEQ ID NO:42.

Another aspect of the invention provides a recombinant polypeptidewherein the heavy chain constant region (CH) region comprises an aminoacid sequence at least 99% identical to any one of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 37 or SEQ ID NO: 38.

In some embodiments, n=0 and Y1-Y2 comprises a chimeric hinge having theamino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8). In otherembodiments, n=0 and Y1-Y2 comprises a chimeric hinge having the aminoacid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9).

In another embodiment of the invention, the recombinant polypeptide isan antigen-binding protein. In another embodiment, the recombinantpolypeptide is a Fc-fusion protein, such as a receptor-Fc fusionprotein. In yet another embodiment of the invention, the recombinantpolypeptide is an antibody.

A further aspect of the invention provides a recombinant polypeptide,antigen-binding protein, Fc-fusion protein or antibody that exhibitsdecreased effector functions when compared to a correspondingrecombinant polypeptide, antigen-binding protein, Fc-fusion protein orantibody comprising the wild-type IgG1 or IgG4 heavy chain constantregion, at a concentration of at least 10 nM. The invention thusprovides a recombinant polypeptide, antigen-binding protein, Fc-fusionprotein or antibody having decreased binding, cytotoxic activity, andcellular proliferation.

The invention further provides a recombinant polypeptide,antigen-binding protein, Fc-fusion protein or antibody that exhibitscytotoxic activity of less than about 50%, at a concentration of atleast 10 nM or at least 100 nM. The invention also provides arecombinant polypeptide, antigen-binding protein, Fc-fusion protein orantibody that exhibits cytotoxic activity of less than about 40%, orless than about 30%, or less than about 20%, or less than about 10%, orless than about 5%, or even undetectable, at a concentration of at least10 nM or at least 100 nM.

In other embodiments, the recombinant polypeptide, antigen-bindingprotein, Fc-fusion protein or antibody exhibits CDC activity of lessthan about 50% cytotoxicity, or less than

40%, 30%, 20%, 10%, or 5%, 4%, 3%, 2%, or even 0% or undetectablecytotoxicity, as measured in an in vitro or ex vivo cell killing assay.In certain embodiments, CDC activity is less than 50%, 40%, 30%, 20%,10%, or 5%, 4%, 3%, 2%, or even 0% or undetectable, at a concentrationof 100 nM. In more embodiments, the recombinant polypeptide,antigen-binding protein, Fc-fusion protein or antibody exhibits ADCCactivity of less than about 50% cytotoxicity, or less than cytotoxicity40%, 30%, 20%, 10%, or 5%, 4%, 3%, 2%, or even 0% or undetectablecytotoxicity, as measured in an in vitro or ex vivo cell killing assay.In certain embodiments, ADCC activity is less than 50%, 40%, 30%, 20%,10%, or 5%, 4%, 3%, 2%, or even 0% or undetectable ADCC activity, at aconcentration of 100 nM.

In still other embodiments, the recombinant polypeptide, antigen-bindingprotein, Fc-fusion protein or antibody exhibits ADCP activity of lessthan about 50% ADCP activity, or less than 40%, 30%, 20%, 10%, or 5%,4%, 3%, 2%, or even 0% or undetectable ADCP activity, as measured in anin vitro or ex vivo cellular phagocytosis assay. In certain embodiments,ADCP activity is less than 50%, 40%, 30%, 20%, 10%, or 5%, 4%, 3%, 2%,or even 0% or undetectable ADCP activity, at a concentration of 100 nM.

The invention further provides a recombinant polypeptide wherein thecytotoxic activity is at least about 10-fold less than the cytotoxicactivity of a corresponding polypeptide comprising a wild-type IgG1 orwild-type IgG4 heavy chain constant region. The invention also providesa recombinant polypeptide wherein the cytotoxic activity is at leastabout 10-fold less, about 20-filed less, about 50-fold less, or about100-fold less, or about 1000-fold less than the cytotoxic activity of acorresponding polypeptide comprising a wild-type IgG1 or wild-type IgG4heavy chain constant region.

An aspect of the invention provides a composition comprising therecombinant polypeptide.

Another aspect of the invention provides a nucleic acid moleculeencoding any one of the recombinant polypeptides of the invention. Theinvention further provides a nucleic acid molecule encoding arecombinant polypeptide of the invention, wherein the recombinantpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 38 and SEQ ID NO: 37.

The invention further provides a nucleic acid molecule comprising anucleotide sequence having greater than 99% sequence identity to SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 33. The inventionalso provides a nucleic acid molecule comprising a nucleotide sequencehaving greater than 99% sequence identity to SEQ ID NO: 36 or SEQ ID NO:35.

The invention also provides a nucleic acid molecule comprising anucleotide sequence selected the group consisting of SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 36 and SEQ ID NO:35.

An aspect of the invention provides a vector comprising any one of thenucleic acid molecules of the invention. The invention further providesa vector wherein the nucleic acid molecule of the invention isoperatively linked to an expression control sequence suitable forexpression in a host cell. The invention also provides a vector of theinvention wherein the expression control sequence comprises a promoterselected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, UbC,RSV, SL3-3, MMTV, Ubi and HIV LTR. The invention further provides avector of the invention wherein the promoter is a CMV-MIE/TetO orCMV-MIE/Arc hybrid promoter. The invention also provides a vector of theinvention comprising one or more selectable marker genes selected fromthe group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM,npt, kanR and pac.

Another aspect of the invention provides a cell comprising a nucleicacid of the invention. The invention further provides a cell comprisinga vector of the invention.

The invention further provides a cell comprising a nucleic acid of theinvention, wherein the nucleic acid is integrated into the genome of thecell. The invention also provides a cell comprising a nucleic acidencoding a protein expression enhancer. The invention still furtherprovides a cell comprising a nucleic acid encoding an XBP polypeptide.

In an embodiment of the invention, the cell is a eukaryotic cell. In anembodiment of the invention, the cell is an animal cell. In anembodiment of the invention, the cell is a mammalian cell. In anotherembodiment of the invention, the cell is a CHO cell. In an embodiment ofthe invention, the cell is a CHO-K1 cell.

An aspect of the invention provides a method of making an antibodycomprising a chimeric hinge region, said method comprising:

(a) transfecting a host cell with a nucleic acid molecule encoding thelight chain of said antibody, said nucleic acid molecule comprising anucleotide sequence encoding the V_(L) region of a selectedantigen-specific antibody and a nucleotide sequence encoding theconstant C_(L) region of an Ig, wherein said nucleotide sequenceencoding the V_(L) region of a selected antigen-specific antibody andsaid nucleotide sequence encoding the C_(L) region of an Ig are operablylinked together; (b) transfecting the host cell of step (a) with anucleic acid molecule encoding the heavy chain of said antibody, saidnucleic acid molecule comprising a nucleotide sequence encoding theV_(H) region of a selected antigen-specific antibody and a nucleotidesequence encoding a constant C_(H) region of a human Ig, wherein thenucleotide sequence encoding the C_(H) region comprises the nucleotidesequence encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 38 or SEQ ID NO: 37, wherein said nucleotide sequenceencoding the V_(H) region of a selected antigen-specific antibody andsaid nucleotide sequence encoding the C_(H) region of said Ig areoperably linked together; and (c) making said antibody by co-expressingthe nucleic acid molecules of (a) and (b) in said host cell.

A method of making an antibody comprising a chimeric hinge region, saidmethod comprising:

(a) transfecting a host cell with a nucleic acid molecule encoding thelight chain of said antibody, said nucleic acid molecule comprising anucleotide sequence encoding the VL region of a selectedantigen-specific antibody and a nucleotide sequence encoding theconstant CL region of an Ig, wherein said nucleotide sequence encodingthe VL region of a selected antigen-specific antibody and saidnucleotide sequence encoding the CL region of an Ig are operably linkedtogether; (b) transfecting the host cell of step (a) with a nucleic acidmolecule encoding the heavy chain of said antibody, said nucleic acidmolecule comprising a nucleotide sequence encoding the VH region of aselected antigen-specific antibody and a nucleotide sequence encoding aconstant CH region of a human Ig, wherein the nucleotide sequenceencoding the CH region comprises the chimeric hinge nucleotide sequenceencoding SEQ ID NO: 8 or SEQ ID NO: 9, wherein said nucleotide sequenceencoding the VH region of a selected antigen-specific antibody and saidnucleotide sequence encoding the CH region of said Ig are operablylinked together; and (c) making said antibody by co-expressing thenucleic acid molecules of (a) and (b) in said host cell.

In another aspect of the invention, the method of making an antibodyfurther comprises the steps of culturing the host cell of step (b)hereinabove, wherein the antibody is secreted into a cell culturemedium; and isolating the antibody from the cell culture media.

An aspect of the invention provides a method of making a receptor-Fcfusion protein comprising a chimeric hinge region, said methodcomprising:

(a) transfecting a host cell with a nucleic acid molecule encoding saidreceptor-Fc fusion protein, said nucleic acid molecule comprising anucleotide sequence encoding a receptor protein, fused to a nucleotidesequence encoding a constant C_(H) region of a human Ig, wherein thenucleotide sequence encoding the C_(H) region comprises the nucleotidesequence encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 38 or SEQ ID NO: 37, wherein said nucleotide sequenceencoding the receptor protein and said nucleotide sequence encoding theC_(H) region of said Ig are operably linked together; and (b) makingsaid receptor-Fc fusion protein by expressing the nucleic acid moleculeof (a) in said host cell.

In another aspect of the invention, the method of making a receptor-Fcfusion comprises the steps of culturing the host cell of step (b)hereinabove, wherein the receptor-Fc fusion protein is secreted into acell culture medium; and isolating the receptor-Fc fusion protein fromthe cell culture media.

An aspect of the invention provides a method of making a receptor-Fcfusion protein comprising a chimeric hinge region, said methodcomprising:

(a) transfecting a host cell with a nucleic acid molecule encoding saidreceptor-Fc fusion protein, said nucleic acid molecule comprising anucleotide sequence encoding a receptor protein, fused to a nucleotidesequence encoding a constant CH region of a human Ig, wherein thenucleotide sequence encoding the CH region comprises the chimeric hingenucleotide sequence encoding SEQ ID NO: 8 or SEQ ID NO: 9, wherein saidnucleotide sequence encoding the receptor protein and said nucleotidesequence encoding the CH region of said Ig are operably linked together;and (b) making said receptor-Fc fusion protein by expressing the nucleicacid molecule of (a) in said host cell.

In other embodiments, the method of making an antibody comprises:

(a) producing a first cell culture comprising cells expressing a firstheavy chain polypeptide of interest;(b) producing a second cell culture comprising cells expressing a secondheavy chain polypeptide of interest;(c) combining the first and second cell culture, or the supernatantsthereof; and(d) recovering the first and second polypeptides in heterodimeric form;wherein the heavy chains of the first and second heavy chainpolypeptides each comprise an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 38 and SEQ ID NO: 37. In the above methods, the firstcell culture further comprises a first cognate light chain of interestand the second cell culture further comprises a second cognate lightchain of interest, wherein the first and second cognate light chains arecovalently bound to the first and second heavy chain polypeptides andare thus recovered. In other embodiments of the above methods, the heavychains of the first and second heavy chain polypeptides each comprise achimeric hinge amino acid sequence selected from the group consisting ofSEQ ID NO: 8 and SEQ ID NO: 9.

In other embodiments, the invention provides a method of making anantibody comprising a first single chain variable fragment-Fc (scFv-Fc)produced in a first cell culture, and a second scFv-Fc produced in asecond cell culture, wherein the Fc of the first and second scFv-Fc eachcomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38and SEQ ID NO: 37, combining the first and second cell cultures or thesupernatants thereof, and recovering or isolating the first and secondscFv-Fc in heterodimeric form. In some embodiments, the first and secondscFv-Fc are secreted into the cell culture medium (e.g. supernatant),and the method comprises combining the first and second cell culturemedia, and recovering or isolating the heterodimeric protein. In someembodiments of the above methods, the Fc of the first and second scFv-Fceach comprises a chimeric hinge amino acid sequence selected from thegroup consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

In some embodiments of the invention, the host cell is selected from thegroup consisting of CHO, COS, retinal cell, Vero, CV1, 293, MDCK, HaK,BHK, HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi,A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMTcell, tumor cell, a cell line derived from any of the aforementionedcells, and a PER.C6® cell.

Another aspect of the invention provides a bispecific antibodycomprising:

-   (a) a first heavy chain comprising an antigen-binding domain capable    of recognizing and binding to a first target antigen, (b) a second    heavy chain comprising an antigen-binding domain capable of    recognizing and binding to a second target antigen, and (c) a common    light chain antigen-binding domain capable of recognizing and    binding to the first or second target antigen, wherein the heavy    chain of (a) or (b) or both (a) and (b) comprises the heavy chain    constant region comprising the amino acid sequence of SEQ ID NO: 1,    SEQ ID NO:2, SEQ ID NO: 30, or SEQ ID NO: 31, or wherein the heavy    chain of (a) or (b) or both (a) and (b) comprises the chimeric hinge    region comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID    NO: 9.

A further aspect of the invention provides a bispecific antibody of theinvention comprising: (a) a first heavy chain comprising a first heavychain constant region comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:30, or SEQ ID NO: 31, and (b) a second heavy chain comprising a secondheavy chain constant region comprising SEQ ID NO: 38 or SEQ ID NO: 37.

These and other objects, along with advantages and features of theinvention disclosed herein, will be made more apparent from thedescription, drawings, and claims that follow.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the corresponding amino acid numbering conventions for thehinge region of hIgG1, hIgG2 and hIgG4. Amino acid numbering isaccording to the most recently updated IMGT Scientific Chart (IMGT®, theinternational ImMunoGeneTics information System®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5^(th) ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991). (wt=wild-type; - means no correspondingnumber was reported for the particular reference; -- means that nocorresponding amino acid was reported in that position for theparticular reference.)

FIG. 2 illustrates hinge amino acids used in the construction ofchimeric hinge regions and the corresponding amino acid conventions.

FIG. 3. Amino acid sequence of the human IgG1 heavy chain constantregion including CH1, hinge, CH2 and CH3 domains as described as IGHG1in UniProtKB/Swiss-Prot Accn. No. P01857 (SEQ ID NO:13).

FIG. 4. Amino acid sequence of the human IgG2 heavy chain constantregion including CH1, hinge, CH2 and CH3 domains as described as IGHG2in UniProtKB/Swiss-Prot Accn. No. P01859 (SEQ ID NO:14).

FIG. 5. Amino acid sequence of the human IgG4 heavy chain constantregion including CH1, hinge, CH2 and CH3 domains as described as IGHG4in UniProtKB/Swiss-Prot Accn. No. P01861 (SEQ ID NO:15).

FIG. 6. Single-parameter histograms showing antibody binding (% of Max)to antigen on Jurkat cells in a fluorescent binding assay. FIG. 6A:Background fluorescence was measured for control assays, i.e. Jurkatcells incubated with secondary antibody only (sec) and unstained Jurkatcells. FIG. 6B: Comparison of Jurkat cell binding to antibody containinga chimeric hinge heavy chain constant region (Ab 1=sIgG4) vs. binding toantibody containing a corresponding antigen-binding domain and awild-type (wt) IgG4 constant region (Control Ab 2). FIG. 6C: Comparisonof Jurkat cell binding to antibody containing a chimeric hinge heavychain constant region (Ab 2=sIgG1) vs. binding to antibody containing acorresponding antigen-binding domain and a wild-type (wt) IgG1 heavychain constant region (Control Ab 1).

FIG. 7. Dose-response curve depicting chimeric hinge antibody ability tobind U937 cells. Half-maximal concentration (EC₅₀) values with respectto binding (mean fluorescence intensity) are given. (Control antibody1=wt IgG1 C_(H); Antibody 2=sIgG1; Control Ab 2=wt IgG4 C_(H); andAntibody 1=sIgG4.)

FIG. 8. Dose-response curve depicting chimeric hinge antibody lack ofcytotoxicity with respect to U937 cells in the presence of activatedPBMCs (T cells). Half-maximal concentration (EC₅₀) values with respectto % cytotoxicity are reflected. (Control antibody 1=wt IgG1 C_(H);Antibody 1=sIgG4; Control Ab 2=wt IgG4 C_(H); and Antibody 2=sIgG1;Control Ab 3=wt IgG1 C_(H).)

FIG. 9. Dose-response curve depicting the half-maximal concentration(EC₅₀) with respect to cell viability in a PBMC proliferation assay.(Control antibody 1=wt IgG1 C_(H); Antibody 1=sIgG4; Control Ab 2=wtIgG4 C_(H); Antibody 2=sIgG1; Control Ab 3=wt IgG1 C_(H); and Control Ab4=non-specific Ab with wt IgG1 C_(H)).

FIG. 10. Dose-response curves depicting lack of CDC activity withrespect to Daudi (FIG. 10A) and Raji (FIG. 10B) cells in the presence ofantibodies having wild-type or chimeric hinge C_(H) regions. (Controlantibody 5=Bispecific Ab with wt IgG1 C_(H); Antibody 3=sIgG1*; Antibody4=sIgG1 *; IgG1 Isotype Control=nonspecific Ab with wt IgG1 C_(H).)

FIG. 11. Dose-response curves depicting lack of ADCC activity withrespect to Daudi (FIG. 11A) and Raji (FIG. 11B) cells in the presence ofantibodies having wild-type or chimeric hinge C_(H) regions. (Controlantibody 5=Bispecific Ab with wt IgG1 C_(H); Antibody 3=sIgG1*; Antibody4=sIgG1*; IgG1 Isotype Control=nonspecific Ab with wt IgG1 C_(H).)

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention is defined by the claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, particular methods and materialsare now described. All publications mentioned herein are incorporatedherein by reference in their entirety.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) chains and one pair of heavy (H) chains, which may all four beinter-connected by disulfide bonds. The structure of immunoglobulins hasbeen well characterized. See for instance Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavychain typically comprises a heavy chain variable region (abbreviatedherein as V_(H) or VH) and a heavy chain constant region (C_(H) or CH).The “heavy chain constant region”, as used herein, typically comprisesthree domains, C_(H)1, C_(H)2, and C_(H)3, whereas the C_(H)1 and C_(H)2domains are linked by a hinge, or a functional fragment thereof. Eachlight chain typically comprises a light chain variable region(abbreviated herein as V_(L) or VL) and a light chain constant region.There are two types of light chains in humans, and other mammals: kappa(K) chain and lambda (A) chain. The light chain constant regiontypically comprises one domain (C_(L)). The V_(H) and V_(L) regions maybe further subdivided into regions of hypervariability (or hypervariableregions which may be hypervariable in sequence and/or form ofstructurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each V_(H) and V_(L) is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus(N-terminus) to carboxy-terminus (C-terminus) in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.Biol. 196, 901-917 (1987)). Typically, the numbering of amino acidresidues in this region is according to IMGT, Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), or by the EU numberingsystem of Kabat (also known as “EU numbering” or “EU index”), e.g., asin Kabat, E. A. et al. Sequences of Proteins of Immunological interest.5^(th) ed. US Department of Health and Human Services, NIH publicationNo. 91-3242 (1991).

The term “antibody” (Ab) as used herein, refers to an immunoglobulinmolecule, or a derivative thereof, which has the ability to specificallybind to an antigen. The variable regions of the heavy and light chainsof the immunoglobulin molecule contain a binding domain that interactswith an antigen as outlined above under “immunoglobulin”. An antibodymay also be a bispecific antibody, diabody, or similar molecule (see forinstance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies).Further, it has been shown that the antigen-binding function of anantibody may be performed by fragments of a full-length antibody, i.e.“antigen-binding fragments” or “antigen-binding proteins”. As with fullantibody molecules, antigen-binding proteins may be monospecific ormultispecific (e.g., bispecific). Examples of binding molecules orfragments encompassed within the term “antibody” include, but are notlimited to (i) a Fab′ or Fab fragment, a monovalent fragment consistingof the V_(L), V_(H), C_(L) and C_(H)1 domains, or a monovalent antibodyas described in the international patent publication numberWO2007059782; (ii) F(ab′)₂ fragments, bivalent fragments comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting essentially of the V_(H) and C_(H)1 domains; (iv)a Fv fragment consisting essentially of a V_(L) and V_(H) domains, (v) adAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consistsessentially of a V_(H) domain and also called domain antibodies (Holt etal; Trends Biotechnol. 2003 November; 21(11):484-90); (vi) camelid ornanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolated complementarity determining region(CDR).

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or during gene rearrangement or bysomatic mutation in vivo).

A multispecific antigen-binding fragment of an antibody (e.g., abispecific antibody) will typically comprise at least two differentvariable domains, wherein each variable domain is capable ofspecifically binding to a separate antigen or to a different epitope onthe same antigen. Any multispecific antibody format, includingbispecific antibody formats disclosed herein, may be adapted for use inthe context of an antigen-binding fragment of an antibody of the presentinvention using routine techniques available in the art.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they may be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain antibodies or singlechain Fv (scFv), see for instance Bird et al., Science 242, 423-426(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such singlechain antibodies are encompassed within the term “antibody” unlessotherwise noted or clearly indicated by context. Although suchpolypeptides are generally included within the meaning of antibody, theycollectively, and each independently, are unique features of the presentinvention, exhibiting different biological properties and utility. Theseand other antibody fragments and recombinant polypeptides that areuseful in the context of the present invention are discussed furtherherein. It also should be understood that the term antibody, unlessspecified otherwise, also includes polyclonal antibodies, monoclonalantibodies (mAbs), antibody-like polypeptides, such as chimericantibodies and humanized antibodies, and any antibody fragmentsretaining the ability to specifically bind to the antigen(antigen-binding fragments or molecules) provided by any knowntechnique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess any Igisotype or combination thereof. Any scFv may be fused to the heavy chainconstant regions of the invention by known techniques.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-basedbispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig,Quadroma, knobs-into-holes, common light chain (e.g., common light chainwith knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, dualacting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein etal. 2012, mAbs 4:6, 1-11, and references cited therein, for a review ofthe foregoing formats). Bispecific antibodies can also be constructedusing peptide/nucleic acid conjugation, e.g., wherein unnatural aminoacids with orthogonal chemical reactivity are used to generatesite-specific antibody-oligonucleotide conjugates which thenself-assemble into multimeric complexes with defined composition,valency and geometry. (See, e.g., Kazane et al. 2013, J. Am. Chem. Soc.9; 135(1):340-6 [Epub: Dec. 21, 2012]).

Further exemplary multispecific formats can be used in the context ofthe present invention include, without limitation, e.g., involving afirst antigen-binding domain that specifically binds a target molecule,and a second antigen-binding domain that specifically binds aninternalizing effector protein, wherein such second antigen-bindingdomains are capable of activating and internalizing the effectorprotein, e.g. a receptor. (See U.S. Application Publication. No.201310243775A1, published on Sep. 19, 2013, which is incorporated byreference.)

Recombinant proteins may be produced by standard molecular biologytechniques well known to the skilled artisan (see e.g., Sambrook, J., E.F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual,Second Edition, Vols 1, 2, and 3, 1989; Current Protocols in MolecularBiology, Eds. Ausubel et al., Greene Publ. Assoc., Wiley Interscience,NY).

The phrase “complementarity determining regions” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin (Ig) genes. CDRs are regions ofhypervariability that are normally (i.e., in the context of a wild-typeanimal) interspersed within the more conserved framework regions (FRs)in a variable region of a light chain or a heavy chain of e.g., anantibody or a T cell receptor (TCR). A CDR can be encoded by, forexample, a germline sequence or a rearranged or unrearranged sequence,and, for example, by a naive or a mature B cell or a T cell. In somecircumstances (e.g., for a CDR3), CDRs can be encoded by two or moresequences (e.g., germline sequences) that are not contiguous (e.g., inan unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “antigen-binding domain”, as used herein, is the amino acidsequence of a heavy chain or light chain capable of selectivelyrecognizing and binding to antigen with a K_(D) at least in themicromolar range. The antigen-binding domain of the invention includesat least one CDR.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. A “variable domain” includes an amino acid sequence capableof folding into a canonical domain (VH or VL) having a dual beta sheetstructure wherein the beta sheets are connected by a disulfide bondbetween a residue of a first beta sheet and a second beta sheet.

The phrase “heavy chain” or “immunoglobulin (Ig) heavy chain”, as usedherein, includes Ig heavy chain constant region sequence from anyorganism, and unless otherwise specified includes a heavy chain variabledomain. Heavy chain variable domains include three heavy chain CDRs andfour FR regions, unless otherwise specified. Fragments of heavy chainvariable domains include CDRs, or both CDRs and FRs. A typical heavychain constant region has, following the variable domain (fromN-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, and aCH3 domain. A functional fragment of a heavy chain in an antigen-bindingprotein includes a fragment that is capable of specifically recognizingan antigen (e.g., recognizing the antigen with a K_(D) in themicromolar, nanomolar, or picomolar range), that is capable of beingexpressed in and secreted from a cell, and that comprises at least oneCDR. For the purposes of this invention, a functional fragment of aheavy chain constant region includes at least an Fc domain or fragmentthereof.

The phrase “Fc-containing protein” includes antibodies, bispecificantibodies, antibody-binding fragments, trap molecules and otherreceptor-Fc fusion proteins, and other binding proteins that comprise atleast a functional portion of an immunoglobulin CH2 and CH3 region, suchas ligand-Fc fusion proteins. A “functional portion” refers to a CH2 andCH3 region that can bind an Fc receptor e.g., an FcγR, (namely FcγRI(CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), or FcγRIIIb(CD16b)) or FcRn, (the neonatal Fc receptor, which confers to antibodiestheir extended half-life). If the CH2 and CH3 region contains deletions,substitutions, and/or insertions or other modifications that render itunable to bind any Fc receptor, then the CH2 and CH3 region isconsidered to be non-functional. As such, when diminishing oreliminating certain effector functions is desired, any Fc-containingprotein may be engineered to comprise a chimeric heavy chain constantregion or fragment as described herein.

The phrase “Fc-fusion proteins”, and specifically “receptor-Fc fusionproteins” includes recombinant proteins engineered to contain afunctional Fc fragment as described herein. For example a “receptor-Fcfusion protein” includes a chimeric protein comprising an amino acidsequence of a receptor protein fused to an amino acid sequence of an Fcdomain of 1 g. Examples of receptor proteins used in fusion proteins areknown in the art (see e.g. Klinkert, et al. J. Neuroimmunol. 72(2):163-8(1997); Milligan, G., et al. Curr Pharm Des. 10(17):1989-2001 (2004);and Schwache D, and Müller-Newen G, Eur J Cell Biol. 91(6-7):428-34(2012), doi: 10.1016/j.ejcb.2011.07.008. Epub 2011 Sep. 29).

In the context of the present invention, receptor-Fc fusion proteins areencoded by a nucleotide sequence encoding a receptor protein fused to anucleotide sequence of a chimeric heavy chain constant region asdescribed herein. In some embodiments, the nucleotide sequence of thereceptor protein encodes for the ligand-binding domain or theextracellular domain of the receptor. In other embodiments, thenucleotide sequence of the receptor protein encodes for theextracellular domain of the receptor and the transmembrane domain of thereceptor. Receptor-Fc fusion proteins are also exemplified inUS20090137416 A1.

Flow cytometry-based autologous secretion trap (FASTR) methods thatutilize the hFcγRI allow rapid isolation of high expression clonesexpressing or secreting an antibody or receptor-Fc fusion protein of theinvention. (See, e.g., US20090137416 A1, which is herein incorporated byreference.) Such high expression clones may be employed to isolate cellsexpressing proteins comprising a chimeric heavy chain constant region ofthe invention. FASTR methods may be utilized to directly screen andisolate cells expressing any recombinant polypeptide, antigen-bindingprotein, antibody, or Fc fusion protein of the invention.

The phrase “light chain” includes an immunoglobulin light chain constantregion sequence from any organism, and unless otherwise specifiedincludes human kappa and lambda light chains. Light chain variable (VL)domains typically include three light chain CDRs and four framework (FR)regions. Generally (i.e. in the context of a wild-type animal), afull-length light chain includes, from amino terminus to carboxylterminus, a VL domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, anda light chain constant domain.

The polypeptides of the invention comprise amino acid sequences that arederived from an immunoglobulin domain. A polypeptide or amino acidsequence “derived from” a designated protein refers to the origin of thepolypeptide.

The term “hinge”, as used herein, is intended to include the region ofconsecutive amino acid residues that connect the C-terminus of theC_(H)1 to the N-terminus of the C_(H)2 domain of an immunoglobulin.Several amino acids of the N-terminus of the C_(H)2 domain, which arecoded by the C_(H)2 exon, are also considered part of the “lower hinge”.Without being bound by any one theory, amino acids of the hinge regionof IgG1, IgG2 and IgG4 have been characterized as comprising 12-15consecutive amino acids encoded by a distinct hinge exon, and severalN-terminal amino acids of the C_(H)2 domain (encoded by the C_(H)2 exon)(Brekke, O. H., et al. Immunology Today 16(2):85-90 (1995)). On theother hand, IgG3 comprises a hinge region consisting of four segments:one upper segment resembling the hinge region of IgG1, and 3 segmentsthat are identical amino acid repeats unique to IgG3.

In the present disclosure for the convenience of the practitioner of theinvention, amino acids of the hinge region for human IgG1, IgG2 and IgG4have been identified herein by the EU numbering system of Kabat (Kabat,E. A. et al., Sequences of Proteins of Immunological interest. 5^(th)ed. US Department of Health and Human Services, NIH publication No.91-3242 (1991)), also known as “EU numbering” or the “EU index”, asupdated according to the IMGT® Scientific Chart, IMGT®, theinternational ImMunoGeneTics information System®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html,created: 17 May 2001, last updated: 10 Jan. 2013.

Correspondence between EU numbering for human IgG1, IgG2 and IgG4 hingeamino acids and IMGT unique domain numbering, IMGT exon numbering, andKabat numbering conventions (see also Kabat, E. A. et al., 1991, supra)are described in FIG. 1 and as follows:

TABLE 1 IgG1 hinge numbering IgG1 (IGHG1) amino acids IMGT Unique[SwissProt Numbering for IMGT Exon EU Kabat P01857] the HINGE^(a)Numbering^(a) Numbering Numbering (E) 1 1 216 226 P 2 2 217 227 K 3 3218 228 S 4 4 219 232^(a) [229]^(b) C 5 5 220 233^(a) [230]^(b) D 6 6221 234^(a) [232]^(b) K 7 7 222 235 T 8 8 223 236 H 9 9 224 237 T 10 10225 238 C 11 11 226 239 P 12 12 227 240 P 13 13 228 241 C 14 14 229 242P 15 15 230 243

TABLE 2 IgG1 C-domain hinge numbering IMGT IgG1 (IGHG1) Unique aminoacids Numbering [SwissProt for IMGT Exon EU Kabat P01857] C-domains^(a)Numbering^(a) Numbering Numbering (A) 1.6 1 231 244 P 1.5 2 232 245 E1.4 3 233 246 L 1.3 4 234 247 L 1.2 5 235 248 G 1.1 6 236 249

TABLE 3 IgG2 hinge numbering IgG2 (IGHG2) IMGT amino Unique acidsNumbering [SwissProt for IMGT Exon EU Kabat P01859] the HINGE^(a)Numbering^(a) Numbering Numbering (E) 1 1 216 226 R 2 2 217 227 K 3 3218 228 C 4 4 219^(a) (221)^(b) 232 C 5 5 220^(a) (—)^(b) 233 V 6 6 222235 E 7 7 224 237 C 8 8 226 239 P 9 9 227 240 P 10 10 228 241 C 11 11229 242 P 12 12 230 243

TABLE 4 IgG2 C-domain hinge numbering IgG2 (IGHG2) amino acids EU Kabat[SwissProt P01859] numbering numbering (A) 231 244 P 232 245 P 233 246 V234 247 A 235 248 -- 236 249

TABLE 5 IgG4 hinge numbering IgG4 (IGHG4) amino acids IMGT Unique[SwissProt Numbering for IMGT Exon EU Kabat P01861] the HINGE^(a)Numbering^(a) Numbering Numbering (E) 1 1 216 226 S 2 2 217 227 K 3 3218 228 Y 4 4 —^(a) (219)^(b) 229 G 5 5 —^(a) (220)^(b) 230 P 6 6 224237 P 7 7 225 238 C 8 8 226 239 P 9 9 227 240 S 10 10 228 241 C 11 11229 242 P 12 12 230 243

TABLE 6 IgG4 C-domain hinge numbering IgG4 (IGHG4) amino acids Kabat[SwissProt P01861] EU Numbering Numbering (A) 231 244 P 232 245 E 233246 F 234 247 L 235 248 G 236 249 Amino acids resulting from exonsplicing are shown in parentheses. — means no corresponding numberreported -- means no corresponding amino acid in this position^(a)numbering according to the last updated IMGT Scientific Chart^(b)numbering according to EU index as reported in Kabat, EA, et al.1991 See also, e.g., Lefranc, M.-P. et al., Devel Comp Immunol, 29,185-203 (2005); and Edelman, G. M. et al. PNAS USA, 63: 78-85 (1969).

For the purposes of this disclosure, an “upper hinge” region is intendedto include amino acid residues from positions 216 to 227 according to EUnumbering (amino acid residues from positions 226 to 240 according toKabat numbering) (see also FIG. 2). A “lower hinge” region is intendedto include amino acid residues from positions 228 to 236 according to EUnumbering (amino acid residues from positions 241 to 249 according toKabat numbering) (see also FIG. 2).

The term “chimeric”, as used herein, means composed of parts ofdifferent origin. The phrase “chimeric protein” includes a first aminoacid protein linked to a second amino acid protein that is not normallylinked in nature. The amino acid sequences may normally exist asseparate proteins or in a different arrangement on the same protein, andare brought together in a fusion polypeptide in a new arrangement.Chimeric proteins may be created by various methods known in the art,e.g. by chemical synthesis or by creating a polynucleotide that encodesfor amino acids of the chimeric protein in the desired arrangement.Exemplary chimeric proteins include the chimeric hinge sequencesconnecting heavy chain domains of IgG, and the fusion proteinsengineered to make the human antibodies, antigen-binding proteins andreceptor-Fc fusion proteins of the present invention.

The chimeric proteins disclosed herein were designed to minimize thecreation of immunogenic epitopes in the junctions, e.g. compared to awild-type IgG Fc region or domain. The engineered proteins of theinvention therefore have reduced immunogenicity, and display reducedbinding to Fc receptors, as well as reduced to no effector functions.

The term “chimeric hinge”, as used herein, is intended to include achimeric protein comprising a first amino acid sequence derived from thehinge region of one Ig molecule and a second amino acid sequence derivedfrom the hinge region of a different class or subclass of Ig molecule.Exemplary chimeric hinges of the present invention comprise a firstamino acid sequence, or an “upper hinge” sequence, derived from a humanIgG1 or human IgG4 hinge region, and a second amino acid sequence, or a“lower hinge” sequence, derived from a human IgG2 hinge region. Incertain embodiments, the first or “upper hinge” sequence comprises aminoacid residues from positions 216 to 227 according to EU numbering. Insome embodiments, the second or “lower hinge” sequence comprises aminoacid residues from positions 228 to 236 according to EU numbering.

The term “humanized antibody”, as used herein, is intended to includeantibodies in which CDR sequences are derived from the germline ofanother mammalian species, such as a mouse, and have been grafted ontohuman framework sequences. Humanized monoclonal antibodies may begenerated by a hybridoma which includes a B lymphocyte cell obtainedfrom a transgenic or transchromosomal nonhuman animal, such as atransgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene, fused to an immortalized cell.For example, when non-human antibodies are prepared with respect to aparticular antigen, the variable regions can be “reshaped” or“humanized” by grafting CDRs derived from nonhuman antibody on the FRspresent in the human antibody to be modified. Application of thisapproach has been reported by Sato, K. et al. Cancer Research 53:851-856(1993); Riechmann, L., et al., Nature 332:323-327 (1988); Verhoeyen, M.,et al., Science 239:1534-1536 (1988); Kettleborough, C. A., et al.,Protein Engineering 4:773-3783 (1991); Maeda, H., et al., HumanAntibodies Hybridoma 2:124-134 (1991); Gorman, S. D., et al., Proc NatlAcad Sci USA 88:4181-4185 (1991); Tempest, P. R., et al., Bio/Technology9:266-271 (1991); Co, M. S., et al., Proc Natl Aced Sci USA 88:2869-2873(1991); Carter, P., et al., Proc Natl Acad Sci USA 89:4285-4289 (1992);and Co, M. S. et al., J Immunol 148:1149-1154 (1992). In someembodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, or six) which are alteredwith respect to the original antibody, which are also termed one or moreCDRs “derived from” one or more CDRs from the original antibody.Humanized antibodies may refer to chimeric molecules prepared usingrecombinant techniques.

In another embodiment, chimeric antibodies comprising human variableregions linked to murine constant regions, such as those produced bycell lines generated by a VELOCIMMUNE mouse, are humanized by replacingthe murine constant region for a human constant region comprising SEQ IDNO: 1, SEQ ID NO:2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38 or SEQID NO:37.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “mouse or murine monoclonal antibody” refers toantibodies displaying a single binding specificity which have variableand constant regions derived from murine or mouse germlineimmunoglobulin sequences.

As used herein, the term “binding” in the context of the binding of anantibody, Ig, antibody-binding fragment, or Fc-containing protein toeither, e.g., a predetermined antigen or to a FcR, typically refers toan interaction or association between a minimum of two entities, ormolecular structures, such as an antibody-antigen interaction, or anFc-containing protein to an FcγR (wherein the Fc-containing protein isan antibody, Ig, antibody-binding fragment, or Fc-fusion protein, e.g.receptor-Fc fusion).

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore 3000 instrument using the antigen or FcRas the ligand and the antibody, Ig, antibody-binding fragment, orFc-containing protein as the analyte (or antiligand). Accordingly, theantibody or other binding protein binds to the predetermined antigen orreceptor with an affinity corresponding to a K_(D) value that is atleast ten-fold lower, such as at least 100 fold lower, for instance atleast 1,000 fold lower, such as at least 10,000 fold lower, for instanceat least 100,000 fold lower than its affinity for binding to anon-specific antigen (e.g., BSA, casein).

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction, orthe dissociation equilibrium constant of an antibody, Ig,antibody-binding fragment, or Fc-containing protein to an FcγR. There isan inverse relationship between K_(D) and binding affinity, thereforethe smaller the K_(D) value, the higher the affinity. Thus, the term“lower affinity” relates to a lower ability to form an interaction andtherefore a larger K_(D) value.

The term “k_(d)” (sec⁻¹ or 1/s), as used herein, refers to thedissociation rate constant of a particular antibody-antigen interaction,or the dissociation rate constant of an antibody, Ig, antibody-bindingfragment, or Fc-containing protein to an FcγR. Said value is alsoreferred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹ or 1/M), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction,or the association rate constant of an antibody, Ig, antibody-bindingfragment, or Fc-containing protein to an FcγR.

The term “K_(A)” (M⁻¹ or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction, orthe association equilibrium constant of an antibody, Ig,antibody-binding fragment, or Fc-containing protein to an FcγR. Theassociation equilibrium constant is obtained by dividing the k_(a) bythe k_(d).

The term “EC₅₀” or “EC50”, as used herein, refers to the half maximaleffective concentration, which includes the concentration of an antibodywhich induces a response halfway between the baseline and maximum aftera specified exposure time. The EC₅₀ essentially represents theconcentration of an antibody where 50% of its maximal effect isobserved. Thus, reduced binding is observed with an increased EC₅₀, orhalf maximal effective concentration value.

In one embodiment, decreased binding can be defined as an increased EC₅₀antibody concentration which enables binding to the half-maximal amountof target cells.

In some embodiments, decreased cytotoxic activity can be defined as anincreased EC₅₀ antibody concentration which enables lysis of thehalf-maximal amount of target cells.

In other embodiments, decreased proliferation can be defined as anincreased EC₅₀ antibody concentration which enables proliferation of thehalf-maximal amount of target cells.

The phrase “bispecific antibody” as used herein includes antibodies thatare specific for different epitopes of one target polypeptide or maycontain antigen-binding domains specific for more than one targetpolypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69. Forexample, the human antibodies can be linked to or co-expressed withanother functional molecule, e.g., another peptide or protein. Forexample, an antibody or fragment thereof can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody or antibody fragment, to produce a bispecific or amultispecific antibody with a second binding specificity. As such,bispecific antibody also includes two antibodies of differentspecificity.

Another strategy, for example using a common light chain, may beemployed as described in US Patent Application Publication No.20100331527A1, wherein two antibodies of different specificity use thesame light chain. In certain embodiments, the heavy chain of at leastone of the Ig heavy chains in a bispecific antibody is modified tocomprise a chimeric heavy chain constant region comprising a recombinantpolypeptide of the invention. In some embodiments, at least one of theheavy chains is modified in the CH3 domain resulting in a differentialaffinity for the bispecific antibody for an affinity reagent, such asProtein A, for ease of isolation. In another embodiment, at least one ofthe heavy chains in such bispecific antibody comprises an amino acidmodification at i) 95R or ii) 95R and 96F in the IMGT numbering system(95R and 96F correspond to 435R and 436F in the EU numbering system),for example SEQ ID NO: 37 and SEQ ID NO: 38. (See US20100331527A1, thecontents of which are herein incorporated by reference.) A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format may be adaptedfor use in the context of an antigen-binding protein or antibody of thepresent invention using routine techniques available in the art.

As used herein, “isotype” refers to the immunoglobulin class (forinstance, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former, but notthe latter, is lost in the presence of denaturing solvents. The epitopemay comprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specific antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specific antigen binding peptide).

As used herein, a humanized antibody is “derived from” a particulargermline sequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody Vdomain sequence is at least 90%, such as at least 95%, for instance atleast 96%, such as at least 97%, for instance at least 98%, or such asat least 99% identical in amino acid V domain sequence to the amino acidsequence encoded by the germline immunoglobulin gene.

Typically, outside the heavy chain CDR3, a human antibody derived from aparticular human germline sequence will display no more than 20 aminoacid differences, e.g. no more than 10 amino acid differences, such asno more than 9, 8, 7, 6 or 5, for instance no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. Mice that express antibodies that are fullyhuman, or partly human and partly mouse, have previously been reported.For example, a transgenic mouse can have a human light chain transgeneand either a human heavy chain transgene or human heavy chaintranschromosome, such that the mouse produces human antibody whenimmunized with target antigen and/or cells expressing the targetantigen. The human heavy chain transgene may be integrated into thechromosomal DNA of the mouse, as is the case for transgenic mice, forinstance HuMAb mice, such as HCo7 or HCol2 mice, or the human heavychain transgene may be maintained extrachromosomally, as is the case fortranschromosomal KM mice as described in WO02/43478. Such transgenic andtranschromosomal mice (collectively referred to herein as “transgenicmice”) are capable of producing multiple isotypes of human monoclonalantibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) byundergoing V-D-J recombination and isotype switching.

VELOCIMMUNE® genetically engineered mice comprise a replacement ofunrearranged V(D)J gene segments at endogenous mouse loci with humanV(D)J gene segments. VELOCIMMUNE® mice express chimeric antibodieshaving human variable domains and mouse constant domains (see, e.g.,U.S. Pat. No. 7,605,237). Most other reports concern mice that expressfully human antibodies from fully human transgenes in mice that havedisabled endogenous immunoglobulin loci.

The VELOCIMMUNE® mouse includes, in part, having a genome comprisinghuman variable regions operably linked to endogenous mouse constantregion loci such that the mouse produces antibodies comprising a humanheavy chain variable region and a mouse heavy chain constant region inresponse to antigenic stimulation. The DNA encoding the variable regionsof the heavy chains of the antibodies can be isolated and operablylinked to DNA encoding the human heavy chain constant regions of theinvention. The DNA can then be expressed in a cell capable of expressingthe fully human heavy chain of the antibody.

A transgenic, nonhuman animal can also be used for production ofantibodies against a specific antigen by introducing into the animalgenes encoding such specific antibody, for example by operativelylinking the genes to a gene which is expressed in the milk of theanimal.

The phrase “effector functions”, as used herein, is intended to includethe functional capabilities imparted by an Fc-containing protein uponbinding to an FcγR. Without being bound to any one theory, formation ofan Fc/FcγR complex recruits a variety of effector cells to sites ofbound antigen, typically resulting in diverse signaling events withinthe cells and important subsequent immune responses.

An “effectorless polypeptide” refers to a recombinant polypeptide,antigen-binding protein or antibody which has altered or reducedeffector function as compared to a corresponding recombinantpolypeptide, antigen-binding protein or antibody comprising a wild-typeheavy chain constant region of the IgG1 or IgG4 isotype. In someembodiments, the effector function that is reduced or altered is acytotoxic effector function, e.g., cytotoxicity, complement-dependentcytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), orantibody-dependent cellular phagocytosis (ADCP). In one embodiment, theeffector function that is reduced or altered is complement-dependentcytotoxicity. In another embodiment, the effector function that isreduced or altered is antibody-dependent cytotoxicity. In otherembodiments, the effector function that is reduced or altered iscellular proliferation.

Several antibody effector functions are mediated at least in part by Fcreceptors (FcRs), which bind the Fc region of an antibody in theconstant domain (specifically, the C_(H)2 and C_(H)3 domain) of atypical immunoglobulin. There are a number of Fc receptors which arespecific for the different classes of immunoglobulins, i.e. IgG, IgE,IgA, IgM, and IgD. The human IgG Fc receptor family is divided intothree groups: FcγRI (CD64), which is capable of binding IgG with highaffinity, FcγRII (CD32) and FcγRIII (CD16) both of which are lowaffinity receptors. Each FcγR subclass is encoded by two or three genes,and alternative RNA splicing leads to multiple transcripts, hence, abroad diversity in FcγR isoforms exists (e.g. FcγRIA (CD64; FCGR1A),FcγRIB (CD64; FCRG1B), FcγRIIA (CD32; FCGR2A), FcγRIIB (CD32; FCGR2B),FcγRIIC (CD32; FCGR2C), FcγRIIIA (CD16a; FCGR3A), and FcγRIIIB (CD16b;FCGR3B)). Additionally, the FcRn, or neonatal Fc receptor (also known asthe Fc receptor transporter, alpha, or FCGRT) is capable of transferringIgG antibodies from mother to fetus across the placenta. Furthermore, Fcreceptors are expressed on a variety of cells, including, e.g., B cells,monocytes, dendritic cells, neutrophils, and certain lymphocytes. Forexample, U937 cells, a human monocyte cell line, express both FcγRI andFcγRIIA (see e.g., Jones, et al. J Immunol 135(5):3348-53 (1985); andBrooks, et al. J Exp Med 170:1369-85 (October 1989)).

Binding of an Ig Fc to its receptor brings these effector cells to sitesof the bound antigen, resulting ultimately in a variety of signaling andimmune responses, including B cell activation, inflammatory responses,cytotoxic responses, and phagocytic responses. As such, reduced oraltered binding of an Ig Fc to its receptor may result in reducedeffector functions.

Alternatively, increased “effector functions” such as cytotoxicity,complement-dependent cytotoxicity (“CDC”), antibody-dependentcytotoxicity (“ADCC”) and abnormal antibody production, may be unwantedside effects associated with certain therapeutic antibodies.

The phrase “antibody-dependent cellular cytotoxicity”,“Antibody-dependent cell-dependent cytotoxicity”, or “ADCC” means anactivity to damage a target cell when an Fcγ receptor-bearing cell (animmune cell or the like) binds to an Fc portion of a specific antibodythrough the Fcγ receptor, when the specific antibody has attached to acell-surface antigen of the target cell. Thus, ADCC is a mechanism bywhich Fc receptor-positive effector cells can lyse target cells thathave adherent antigen-specific molecule. The ADCC activity can beevaluated by a number of well-known methods, including measuring thefluorescent intensity using a fluorescent dye such as calcein AM (WakoPure Chemical Industries, Ltd., 349-07201). When this approach isemployed, the cytotoxic activity (%) can be calculated, using theobtained values, according to the equation: (A−C)/(B−C)×100, wherein Ais a fluorescent value in each sample, B is an average fluorescent valueof the cells lysed and released into a medium with Nonidet P-40 having afinal concentration of 1%, and C is an average fluorescent value whenonly the medium was added.

The phrase “antibody-dependent cellular phagocytosis” or “ADCP”, as usedherein, relates to effector function that eliminates (or kills) a targetcell by engulfing the target cell rather than inducing cytolysis. ADCPmay be an important in vivo mechanism for killing tumor cells. ADCP canbe measured by two-color fluorescence flow cytometry methods, forexample methods utilizing, e.g. PKH2 (green fluorescent dye) andphycoerythrin-conjugated (red) monoclonal antibodies against differentcell surface proteins to differentiate the test cells, thus determiningphagocytic activity and rate of phagocytosis. Therapeutic strategiesthat selectively activate FcγRIIa relative to FcγRIIb may enhancemacrophage phagocytic activity (Richards, J O, et al. 2008 Mol CancerTher 7(8):2517-27).

The phrase “complement-directed cytotoxicity” or “CDC”, as used herein,includes a cytotoxic activity by the complement system. The CDC activityis measured by well-known methods, for example the target cells,antibody, and complement solution (such as baby rabbit complement(Cedarlane Technologies) are incubated and are allowed to react,according to standard protocols (NIAID Manual of Tissue TypingTechniques 1979-1980, Edited by J. G. Ray, NIH Publication No.NIH-83-545.) The cytotoxic activity can be calculated in the same manneras the measurement of the ADCC activity. The cytotoxic activity can alsobe measured using a fluorescent dye (such as calcein) or radioactivedyes similarly to the above with respect to ADCC.

The phrase “cytotoxicity” or “direct cytotoxicity” includes anycytotoxic activity including that which is independent of NK cells.Cytotoxicity may be measured by techniques well known in the art, forexample, determining cell lysis or cell death, i.e. apoptosis. Directcell lysis, or cell killing, can be evaluated by a number of well-knownmethods, including measuring the fluorescent intensity using calcein andcalculating an average fluorescent value in a similar fashion asdescribed with respect to ADCC hereinabove. Alternatively, a cytotoxicmolecule, such as an antibody, is effective in an apoptosis assay byactivating a genetic program of controlled cell death. Apoptosis ischaracterized by well defined cytological and molecular events includinga change in the refractive index of the cell, cytoplasmic shrinkage,nuclear condensation and cleavage of DNA into regularly sized fragments.Cells that are undergoing apoptosis shut down metabolism, lose membraneintegrity and form membrane blebs. The apoptotic activity is measuredusing standard methods of determining dying and/or dead cells. In orderto measure apoptosis, cytotoxicity assays can be employed. These assaysmay be radioactive or non-radioactive assays that measure increases inplasma membrane permeability, since dying cells become leaky,colorimetric assays may be employed that measure reduction in themetabolic activity of mitochondria based on the knowledge thatmitochondria in dead cells cannot metabolize dyes, while mitochondria inlive cells can. Bioluminescent cytotoxicity assays (e.g. CytoTox-Glo™,Promega) were developed to measure the release of stable proteasemarkers into the cell culture medium. Protease activity is considered arobust enzymatic cell death marker, and may be used as a ratiometricmeasurement of viable and dead cells (Niles, A. L., et al. Anal Biochem,366(2): 197-206, 15 Jul. 2007).

One can also measure early indicators for apoptosis such as alterationsin membrane asymmetry resulting in occurrence of phosphatidylserine onthe outside of the cell surface (Annexin V based assays). Alternatively,later stages of apoptosis, such as activation of caspases can bemeasured in populations of cells or in individual cells. In addition,measurement of release of cytochrome C and AIF into cytoplasm bymitochondria or fragmentation of chromosomal DNA can be determined.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) isa common method for detecting DNA fragmentation that results fromapoptotic signaling cascades. The assay relies on the presence of nicksin the DNA which can be identified by terminal deoxynucleotidyltransferase, an enzyme that will catalyze the addition of bromolateddUTPs that are secondarily detected with a specific labelled antibody.

Cytotoxicity may be complement-directed, or antibody-directed, ordirectly associated with the binding of a cytotoxic molecule or cell.

In certain embodiments, antibodies of the invention exhibit cytotoxicityof less than 20% cytolysis (i.e. % cytotoxicity), or less than 10%, or5%, 4%, 3%, 2%, or even 0% or undetectable cytolysis (cytotoxicity), asmeasured in an in vitro or ex vivo cell killing assay. In certainembodiments, antibodies of the invention exhibit less than 20%, 10%, or5%, 4%, 3%, 2%, or even 0% or undetectable cytotoxicity, at an antibodyconcentration of 10 nM.

In other embodiments, antibodies exhibit apoptotic activity of less than20% cytolysis (i.e. % cytotoxicity), or less than 10%, or 5%, 4%, 3%,2%, or even 0% or undetectable cytolysis (cytotoxicity), as measured inan in vitro or ex vivo cell killing assay. In still other embodiments,In certain embodiments, antibodies of the invention exhibit less than20%, 10%, or 5%, 4%, 3%, 2%, or even 0% or undetectable apoptoticactivity, at an antibody concentration of 10 nM.

In still other embodiments, antibodies exhibit CDC activity of less thanabout 50% cytotoxicity, or less than cytotoxicity 40%, 30%, 20%, 10%, or5%, 4%, 3%, 2%, or even 0% or undetectable cytotoxicity, as measured inan in vitro or ex vivo cell killing assay. In still other embodiments,In certain embodiments, antibodies of the invention exhibit less than50%, 40%, 30%, 20%, 10%, or 5%, 4%, 3%, 2%, or even 0% or undetectableCDC activity, at an antibody concentration of 100 nM. In moreembodiments, antibodies exhibit ADCC activity of less than about 50%cytotoxicity, or less than cytotoxicity 40%, 30%, 20%, 10%, or 5%, 4%,3%, 2%, or even 0% or undetectable cytotoxicity, as measured in an invitro or ex vivo cell killing assay. In still other embodiments, Incertain embodiments, antibodies of the invention exhibit less than 50%,40%, 30%, 20%, 10%, or 5%, 4%, 3%, 2%, or even 0% or undetectable ADCCactivity, at an antibody concentration of 100 nM.

The present invention provides antibodies, antigen-binding proteins andFc-fusion proteins that comprise recombinant polypeptides comprising achimeric hinge, and further provide reduced effector functions. Theproperties of such recombinant polypeptides of the invention may becompared to the properties of one or more reference proteins. See theexamples below for reference, or control antibodies and antigen-bindingproteins which have corresponding variable regions and constant regions(e.g. having a wild-type IgG1 C_(H) region (SEQ ID NO:13) or a wild-typeIgG4 C_(H) region (SEQ ID NO:15)) compared to the chimeric antibodies ofthe invention, and may be used in certain testing methodologies forcomparison of functional or pharmacokinetic properties to the antibodiesand antigen-binding proteins of the invention. It is understood that acorresponding wild-type IgG C_(H) region differs from chimeric C_(H)regions of the invention in that the “wild-type” IgG C_(H) region isderived from the natural IgG amino acid sequence containing (fromN-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3domain. Wild-type IgG may include variants having one or more amino acidmodifications while retaining essentially the same functionalcharacteristics as natural IgG. A corresponding antibody or polypeptidemay be assembled to have the same target binding domain (e.g. V_(H)and/or V_(L)) as the antibody or polypeptide having the chimeric C_(H)region, or otherwise has essentially the same functionality for thepurpose of comparison in certain assays.

It has been shown in the Examples that isolated antibodies of theinvention bind weakly to FcγR expressing cells, e.g. effector cells, buthowever lack effector functions, such as cytotoxicity and proliferation,compared to the effector functions of a corresponding antibodycomprising a wild-type IgG1 or IgG4 C_(H) region.

FURTHER ASPECTS OF THE INVENTION

In one aspect, the invention provides a recombinant polypeptidecomprising a chimeric hinge region. In some aspects, the chimeric hingeregion comprises a human IgG2 lower hinge amino acid sequence PCPAPPVA(SEQ ID NO: 3) from positions 228 to 236 (EU numbering). In otheraspects, the chimeric hinge region comprises: a human IgG1 or a humanIgG4 upper hinge amino acid sequence from positions 216 to 227 (EUnumbering) and a human IgG2 lower hinge amino acid sequence PCPAPPVA(SEQ ID NO: 3) from positions 228 to 236 (EU numbering), from N-terminusto C-terminus.

In other aspects, the chimeric hinge region comprises: an upper hingeamino acid sequence from positions 216 to 227 (EU numbering) selectedfrom the group consisting of SEQ ID NO:4 and SEQ ID NO:5, and a lowerhinge amino acid sequence PCPAPPVA (SEQ ID NO: 3) from positions 228 to236 (EU numbering), from N-terminus to C-terminus.

The invention provides a recombinant polypeptide comprising, fromN-terminus to C-terminus, a CH1 domain, a chimeric hinge, a CH2 domain,and a CH3 domain wherein: the CH1 domain comprises at least the aminoacid sequence DKKV or DKRV from positions 212 to 215 (EU numbering), thechimeric hinge comprises a human IgG1 or a human IgG4 upper hinge aminoacid sequence from positions 216 to 227 (EU numbering) and a human IgG2lower hinge amino acid sequence PCPAPPVA (SEQ ID NO: 3) from positions228 to 236 (EU numbering), the CH2 domain comprises a human IgG4 CH2domain amino acid sequence from positions 237 to 340 (EU numbering), andthe CH3 domain comprises a human IgG1 or a human IgG4 CH3 domainsequence from positions 341 to 447 (EU numbering), or a variant thereof.In one embodiment, the present invention provides a monoclonal antibodycomprising a heavy chain constant region comprising or consisting of SEQNO: 1, SEQ ID NO: 2, SEQ ID NO: 30, or SEQ ID NO: 31. In anotherembodiment, the present invention provides a monoclonal antibodycomprising at least one heavy chain with a constant region comprising orconsisting of SEQ ID NO: 38 or 37.

TABLE 7 Representative chimeric C_(H )constructs C_(H )construct LowerSEQ ID NO CH1 Upper Hinge Hinge CH2 CH3  1 DKRV ESKYGPPCP PCPAPPVASEQ ID NO: 10 SEQ ID NO: 12 (SEQ ID NO: 5) (SEQ ID NO: 7) (SEQ ID NO: 3) 2 DKKV EPKSCDKTHTCP PCPAPPVA SEQ ID NO: 10 SEQ ID NO: 11 (SEQ ID NO: 4)(SEQ ID NO: 6) (SEQ ID NO: 3) 30 SEQ ID NO: 43 EPKSCDKTHTCP PCPAPPVASEQ ID NO: 10 SEQ ID NO: 11 (SEQ ID NO: 6) (SEQ ID NO: 3) 31SEQ ID NO: 44 ESKYGPPCP PCPAPPVA SEQ ID NO: 10 SEQ ID NO: 12(SEQ ID NO: 7) (SEQ ID NO: 3) 37 SEQ ID NO: 43 EPKSCDKTHTCP PCPAPPVASEQ ID NO: 10 SEQ ID NO: 41 (SEQ ID NO: 6) (SEQ ID NO: 3) 38SEQ ID NO: 44 ESKYGPPCP PCPAPPVA SEQ ID NO: 10 SEQ ID NO: 42(SEQ ID NO: 7) (SEQ ID NO: 3)

In some embodiments, the recombinant polypeptide is selected from thegroup consisting of an antibody, antigen-binding protein and receptor-Fcfusion protein.

In some embodiments, the isolated antibody, antigen-binding protein, orreceptor-Fc fusion protein comprises a heavy chain construct comprisinga CH region, from N-terminus to C-terminus, a CH1 domain, a chimerichinge, a CH2 domain, and a CH3 domain, wherein the CH1 domain comprisesthe amino acid sequence DKKV or DKRV, the chimeric hinge comprises ahuman IgG1 or a human IgG4 upper hinge amino acid sequence frompositions 216 to 227 (EU numbering), or a natural variant thereof, and ahuman IgG2 lower hinge amino acid sequence from positions 228 to 236 (EUnumbering), the CH2 domain comprises a human IgG4 CH2 domain sequencefrom positions 237 to 340 (EU numbering), or a natural variant thereof,and the CH3 domain comprises a human IgG1 or a human IgG4 CH3 domainsequence from positions 341 to 447 (EU numbering), or a natural variantthereof, and the CH region has an amino acid sequence with at leastabout 95%, or about 96%, or about 97%, or about 98%, or about 99%identity to SEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention provides an isolatedantibody, antigen-binding protein, or receptor-Fc fusion proteincomprising a heavy chain construct comprising a CH region, fromN-terminus to C-terminus, a CH1 domain, a chimeric hinge, a CH2 domain,and a CH3 domain, wherein the CH1 domain comprises the amino acidsequence DKKV or DKRV, the chimeric hinge comprises a human IgG1 or ahuman IgG4 upper hinge amino acid sequence from positions 216 to 227 (EUnumbering), or a natural variant thereof, and a human IgG2 lower hingeamino acid sequence from positions 228 to 236 (EU numbering), the CH2domain comprises a human IgG4 CH2 domain sequence from positions 237 to340 (EU numbering), or a natural variant thereof, and the CH3 domaincomprises a human IgG1 or a human IgG4 CH3 domain sequence frompositions 341 to 447 (EU numbering), or a natural variant thereof, andthe CH region has an amino acid sequence with at least about 95%, orabout 96%, or about 97%, or about 98%, or about 99% identity to SEQ IDNO: 30 or SEQ ID NO: 31. In some embodiments, the CH region has an aminoacid sequence with at least about 95%, or about 96%, or about 97%, orabout 98%, or about 99% identity to SEQ ID NO: 38 or SEQ ID NO: 37.

Such “variant” or “natural variant” C_(H) domains of the invention, e.g.Fc domains and Fc domain fragments, comprise one or more additions,deletions, or substitutions of amino acids when compared to thewild-type sequence that such C_(H) domains of the invention are derivedfrom, but essentially function as desired. In some examples, the C_(H)domain or Fc domain exhibits weak or no binding to certain FcγRexpressing cells, e.g. effector cells, resulting in altered effectorfunctions, such as cytotoxicity and proliferation. In one example, suchvariants include modifications such as additions, deletions, orsubstitutions of amino acids in the CH3 domain engineered for theisolation of bispecific molecules.

In one embodiment, the present invention provides a monoclonal antibodycomprising a heavy chain variable region and a heavy chain constantregion comprising or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO: 37, or a sequence withat least about 95%, or about 96%, or about 97%, or about 98%, or about99% to such heavy chain constant region. In another embodiment, thepresent invention provides a monoclonal antibody comprising a lightchain variable region, a heavy chain variable region and a heavy chainconstant region comprising or consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO: 37, or asequence with at least about 95%, or about 96%, or about 97%, or about98%, or about 99% to such heavy chain constant region.

In another embodiment, the invention is an isolated antibody, orantigen-binding fragment thereof, comprising a heavy chain constantregion (C_(H)) having (i) the amino acid sequence of SEQ NO: 1 or havingthe amino acid sequence encoded by SEQ ID NO: 33, or (ii) the amino acidsequence of SEQ NO: 31 or having the amino acid sequence encoded by SEQID NO: 29, or (iii) a sequence with at least about 95%, or about 96%, orabout 97%, or about 98%, or about 99% identity to a sequence describedin (i) or (ii). In another embodiment, the invention is an isolatedantibody, or antigen-binding fragment thereof, comprising a heavy chainconstant region (C_(H)) having (i) the amino acid sequence of SEQ NO: 2or having the amino acid sequence encoded by SEQ ID NO: 32, or (ii) theamino acid sequence of SEQ NO: 30 or having the amino acid sequenceencoded by SEQ ID NO: 28, or (iii) a sequence with at least about 95%,or about 96%, or about 97%, or about 98%, or about 99% identity to asequence described in (i) or (ii).

In still another embodiment, the invention is an isolated antibody, orantigen-binding fragment thereof, comprising a heavy chain constantregion (C_(H)) having (i) the amino acid sequence of SEQ NO: 38 orhaving the amino acid sequence encoded by SEQ ID NO: 36, or (ii) theamino acid sequence of SEQ NO: 37 or having the amino acid sequenceencoded by SEQ ID NO: 35, or (iii) a sequence with at least about 95%,or about 96%, or about 97%, or about 98%, or about 99% identity to asequence described in (i) or (ii).

In some embodiments, the isolated antibody is a monoclonal antibody. Inother embodiments, the isolated antibody, or antigen binding fragmentthereof, is a humanized, chimeric, single-chain antibody or bispecificantibody.

In another embodiment, the invention is an Fc-containing proteincomprising (i) the amino acid sequence of SEQ NO: 1 or the amino acidsequence encoded by SEQ ID NO: 33, or (ii) the amino acid sequence ofSEQ NO: 31 or the amino acid sequence encoded by SEQ ID NO: 29, or (iii)a sequence with at least about 95%, or about 96%, or about 97%, or about98%, or about 99% identity to a sequence described in (i) or (ii). Inanother embodiment, the invention is an Fc-containing protein comprising(i) the amino acid sequence of SEQ NO: 2 or the amino acid sequenceencoded by SEQ ID NO: 32, or (ii) the amino acid sequence of SEQ NO: 30or the amino acid sequence encoded by SEQ ID NO: 28, or (iii) a sequencewith at least about 95%, or about 96%, or about 97%, or about 98%, orabout 99% identity to a sequence described in (i) or (ii).

In another embodiment, the invention is an Fc-containing proteincomprising (i) the amino acid sequence of SEQ NO: 38 or the amino acidsequence encoded by SEQ ID NO: 36, or (ii) the amino acid sequence ofSEQ NO: 37 or the amino acid sequence encoded by SEQ ID NO: 35, or (iii)a sequence with at least about 95%, or about 96%, or about 97%, or about98%, or about 99% identity to a sequence described in (i) or (ii).

In another embodiment, the present invention provides a compositioncomprising the antibody, antigen-binding protein or Fc fusion proteindescribed.

The present invention provides an antibody or antigen-binding proteinthat lacks cytotoxic or cytolytic effector function. In some aspects,the antibody or antigen-binding protein lacks cytotoxic or cytolyticeffector function and comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO: 37.

The present invention provides an antibody or antigen-binding proteinhaving cytotoxic activity that is at least 10-fold less, or at least50-fold less, or at least 100-fold less, or at least 1000-fold less, orat least 10000-fold less than the cytotoxic activity of a correspondingantibody comprising a wild-type IgG1 or wild-type IgG4 CH region.

The present invention provides an antibody capable of binding to anFcγR, wherein such antibody comprises a recombinant polypeptidecomprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 38, or SEQ ID NO: 37. In some embodiments, the antibody iscapable of binding to an FcγR with lower affinity compared to thebinding affinity of a corresponding antibody comprising a wild-type IgG1or wild-type IgG4 heavy chain constant region. In some embodiments, theantibody is capable of activating an FcγR at a half-maximalconcentration (EC₅₀) of greater than about 10 nM, or about 20 nM, orabout 30 nM, or about 40 nM, about 50 nM, about 100 nM. In someembodiments, the FcγR is a human or cynomolgus FcγRII. In otherembodiments, the FcγR is FcγRIIA or FcγRIIB. In some embodiments, theantibody is capable of binding to FcγRIIA having an affinity (K_(D)value) of about 10 μM, or about 20 μM, or about 30 μM, or about 40 μM,about 50 μM, about 100 μM. In other embodiments, the antibody orrecombinant polypeptide binds to FcγRIIA with an affinity greater thanthe affinity of the antibody or recombinant polypeptide to FcγRIIB.

In some embodiments, the antibody is capable of binding to FcγRIIBhaving an affinity (KD value) of about 10 μM, or about 20 μM, or about30 μM, or about 40 μM, about 50 μM, about 100 μM. In some embodiments,the antibody or recombinant polypeptide binds to FcγRIIA with anaffinity (K_(D) value) substantially similar to the affinity of theantibody or recombinant polypeptide to FcγRIIB.

For certain embodiments, it may be desirable for the chimeric antibodiesof the invention to engage, and even indirectly enhance FcγRIIA-mediatedactivity, even though the chimeric antibodies may have wild-typeaffinities for FcγRIIA. Without being bound to any one theory, certainantibodies, and thus therapeutics, may benefit from a weakenedinteraction (compared to wild-type antibodies) with the inhibitoryreceptor FcγRIIB, which may shift the balance between the activatingFcγRIIA and the inhibitory FcγRIIB receptors in favor of activation.

The present invention encompasses the production of monoclonalantibodies comprising a recombinant polypeptide comprising SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO:37 and having specificities for various target antigens. The inventionprovides a method for producing an antibody comprising: a) introducingthe vector comprising a nucleic acid molecule encoding an antibody, orfragment thereof, of the invention into a mammalian host cell, b)culturing the host cell capable of expressing the antibody, and c)isolating the antibody from the cell culture media.

Monoclonal antibodies of the present invention may e.g. be produced bythe hybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature 352,624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B lymphocyte cells obtained from miceimmunized with an antigen of interest, for instance, in the form ofcells expressing the antigen on the surface, or a nucleic acid encodingan antigen of interest. Monoclonal antibodies may also be obtained fromhybridomas derived from antibody-expressing cells of immunized humans ornon-human mammals such as rats, rabbits, dogs, primates, etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies may be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. Such transgenic and transchromosomic mice includemice referred to herein as VELOCIMMUNE mice, HuMAb mice and KM mice,respectively, and are collectively referred to herein as “transgenicmice” and are described hereinabove.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. No. 5,827,690, U.S. Pat. No.5,756,687, U.S. Pat. No. 5,750,172 and U.S. Pat. No. 5,741,957.Expression vectors comprising a mouse UP-II promoter operatively linkedto the DNA encoding the Ig heavy and light chain sequences of interestmay be engineered for expression and secretion of the proteins ofinterest in urine of a transgenic animal (See, e.g., U.S. Pat. No.5,824,543).

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be generated throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, and other techniques, usingtechniques well known in the art and the resulting molecules may besubjected to additional maturation, such as affinity maturation, as suchtechniques are well known in the art (see for instance Hoogenboom etal., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized.

The engineered heavy chain constant (C_(H)) regions of the inventionwill provide reduced effector functions. Either of the human light chainconstant (CO regions, kappa or lambda, may be used. If desired, theclass of an antibody of the present invention may be switched by knownmethods.

The present invention provides the antibodies of the invention producedby a host cell. In one embodiment, the invention provides a method forproducing a monoclonal antibody comprising a) immunizing VELOCIMMUNE®mice with an antigen sufficient to cause an antibody immune response, b)obtaining serum from such mice and testing for antibody titer againstsaid antigen, c) harvesting B cells from the spleens of such immunizedmice shown to have elevated antibody titers and fusing said B cells withmouse myeloma cells to form such hybridoma, d) isolating chimericantibody from such hybridoma by protein A chromatography, such chimericantibody having a human variable region and a mouse constant region, e)selecting a chimeric antibody having desirable characteristics, and f)replacing the mouse constant regions of such antibodies with a humanconstant region of the invention, for example, such human constantregion comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 38, or SEQ ID NO: 37.

In one embodiment, the antibody of the invention is a full-length IgGantibody. In another embodiment, the antibody of the invention is anantibody fragment or a single-chain antibody. In still anotherembodiment, the antibody of the invention is a fully human IgG antibody.

In some aspects of the invention, the antibody is a bispecific antibodywherein each antigen-binding domain of such molecule or antibodycomprises a V_(H) region paired with a V_(L) region. In certainembodiments, the bispecific antibody comprises a first antigen-bindingdomain and a second antigen binding domain each comprise different,distinct V_(H) regions ith a common V_(L) region. In some embodiments,the bispecific antibodies are constructed comprising a firstantigen-binding domain that specifically binds a first antigen, whereinthe first antigen-binding domain comprises an V_(H) region/V_(L) regionpair derived from a first antibody directed against the first antigen;and a second antigen-binding domain that specifically binds a secondantigen, wherein the second antigen-binding domain comprises an V_(H)region derived from a second antibody directed against a second antigenpaired with an V_(L) region derived from the first antibody (e.g., thesame V_(L) region that is included in the antigen-binding domain of thefirst antibody). In some embodiments, the heavy chain of at least one ofthe antibodies, i.e. the first antibody or the second antibody or bothantibodies, in a bispecific antibody is modified to comprise a chimericheavy chain constant region (C_(H) region). In other embodiments, thebispecific antibody comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO: 37.

In some aspects of the invention, two antibodies of differentspecificity use the same light chain. In certain embodiments, the heavychain of at least one of the Ig heavy chains in a bispecific antibody ismodified to comprise a chimeric heavy chain constant region comprising arecombinant polypeptide of the invention. In some embodiments, at leastone of the heavy chains is modified in the CH3 domain resulting in adifferential affinity for the bispecific antibody for an affinityreagent, such as Protein A, for ease of isolation. In anotherembodiment, at least one of the heavy chains in such bispecific antibodycomprises an amino acid modification at i) 95R or ii) 95R and 96F in theIMGT numbering system (95R and 96F correspond to 435R and 436F in the EUnumbering system).

In other aspects, the antibody is a bispecific antibody wherein thebispecific antibody comprises:

(a) a first heavy chain comprising an antigen-binding domain capable ofrecognizing and binding to a first target antigen,(b) a second heavy chain comprising an antigen-binding domain capable ofrecognizing and binding to a second target antigen,(c) a common light chain antigen-binding domain capable of recognizingand binding to the first or second target antigen,wherein the heavy chain of (a) or (b) or both (a) and (b) furthercomprises the heavy chain constant region comprising SEQ ID NO:1 or SEQID NO: 31.

In still other aspects, the antibody is a bispecific antibody whereinthe bispecific antibody comprises:

(a) a first heavy chain comprising an antigen-binding domain capable ofrecognizing and binding to a first target antigen,(b) a second heavy chain comprising an antigen-binding domain capable ofrecognizing and binding to a second target antigen,(c) a common light chain antigen-binding domain capable of recognizingand binding to the first or second target antigen,wherein the heavy chain of (a) or (b) or both (a) and (b) furthercomprises the heavy chain constant region comprising SEQ ID NO:2 or SEQID NO: 30.

In another aspect, at least one of the heavy chains of (a) or (b) insuch bispecific antibody hereinabove comprises an amino acidmodification at (i) 435R or (ii) 435R and 436F (EU numbering) ((i)₉₅R or(ii) 95R and 96F in the IMGT numbering system).

In other aspects, the antibody is a bispecific antibody wherein thebispecific antibody comprises (a) a first heavy chain comprising anantigen-binding domain capable of recognizing and binding to a firsttarget antigen, and a first heavy chain constant region comprising SEQID NO: 1, SEQ ID NO:2, SEQ ID NO: 30, or SEQ ID NO: 31; (b) a secondheavy chain comprising an antigen-binding domain capable of recognizingand binding to a second target antigen, and a second heavy chainconstant region comprising SEQ ID NO: 38 or SEQ ID NO: 37; and (c)common light chain antigen-binding domain capable of recognizing andbinding to the first or second target antigen.

In one embodiment, the antibody is a monovalent antibody. Accordingly,in one embodiment, the antibody is a monovalent antibody, wherein saidantibody is constructed by a method comprising: i) providing a nucleicacid molecule encoding the light chain of said monovalent antibody, saidconstruct comprising a nucleotide sequence encoding the V_(L) region ofa selected antigen specific antibody and a nucleotide sequence encodingthe constant C_(L) region of an Ig, wherein said nucleotide sequenceencoding the V_(L) region of a selected antigen specific antibody andsaid nucleotide sequence encoding the C_(L) region of an Ig are operablylinked together, and wherein the nucleotide sequence encoding the C_(L)region has been modified such that the C_(L) region does not contain anyamino acids capable of forming disulfide bonds or covalent bonds withother peptides comprising an identical amino acid sequence of the C_(L)region in the presence of polyclonal human IgG or when administered toan animal or human being; ii) providing a nucleic acid constructencoding the heavy chain of said monovalent antibody, said constructcomprising a nucleotide sequence encoding the V_(H) region of a selectedantigen specific antibody and a nucleotide sequence encoding a constantC_(H) region of a human Ig, wherein the nucleotide sequence encoding theC_(H) region comprises nucleic acids encoding SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO: 37, whereinsuch nucleotide sequence has been modified and does not comprise anyamino acid residues which participate in the formation of disulphidebonds or covalent or stable non-covalent inter-heavy chain bonds withother peptides comprising an identical amino acid sequence of the C_(H)region of the human Ig in the presence of polyclonal human IgG or whenadministered to an animal, such as a human, wherein said nucleotidesequence encoding the V_(H) region of a selected antigen specificantibody and said nucleotide sequence encoding the C_(H) region of saidIg are operably linked together; iii) providing a cell expression systemfor producing said monovalent antibody; iv) producing said monovalentantibody by co-expressing the nucleic acid constructs of (i) and (ii) incells of the cell expression system of (iii).

Similarly, in one embodiment, the antibody is a monovalent antibody,which comprises: i) a variable region or an antigen-binding domain ofsaid region, and ii) a C_(H) region of an immunoglobulin or a fragmentthereof comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 38, or SEQ ID NO: 37, wherein the C_(H) region orfragment thereof has been modified such that the region does notcomprise any amino acid residues which are capable of forming disulfidebonds with an identical C_(H) region or other covalent or stablenon-covalent inter-heavy chain bonds with an identical C_(H) region inthe presence of polyclonal human IgG.

In another further embodiment, the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

In general, antibodies described herein may be modified by inclusion ofany suitable number of such modified amino acids and/or associationswith conjugated substituents. Suitability in this context is generallydetermined by the ability to at least substantially retain the antibodyor antigen binding fragment's selectivity and/or specificity associated.The modified amino acid may, for instance, be selected from aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, or an amino acid conjugated to an organicderivatizing agent. The inclusion of one or more modified amino acidsmay be advantageous in, for example, further increasing polypeptideserum half-life, reducing polypeptide antigenicity, or increasingpolypeptide storage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N—X—S/T motifs during expression inmammalian cells) or modified by synthetic means. Non-limiting examplesof a modified amino acid include a glycosylated amino acid, a sulfatedamino acid, a prenylated (e.g., farnesylated, geranylgeranylated) aminoacid, an acetylated amino acid, an acylated amino acid, a PEGylatedamino acid, a biotinylated amino acid, a carboxylated amino acid, aphosphorylated amino acid, and the like. References adequate to guideone of skill in the modification of amino acids are replete throughoutthe literature. Example protocols are found in Walker (1998) ProteinProtocols On CD-Rom, Humana Press, Totowa, N.J.

Antibodies of the invention may also be chemically modified by covalentconjugation to a polymer to, for instance, further increase theircirculating half-life. Exemplary polymers, and methods to attach them topeptides, are illustrated in for instance U.S. Pat. No. 4,766,106, U.S.Pat. No. 4,179,337, U.S. Pat. No. 4,495,285 and U.S. Pat. No. 4,609,546.Additional illustrative polymers include polyoxyethylated polyols andpolyethylene glycol (PEG) (e.g., a PEG with a molecular weight ofbetween about 1,000 and about 40,000, such as between about 2,000 andabout 20,000, e.g., about 3,000-12,000 g/mol).

In one embodiment, antibodies comprising one or more radiolabeled aminoacids are provided. A radiolabeled antibody may be used for bothdiagnostic and therapeutic purposes. In another embodiment, antibodiesof the present invention may be conjugated to a molecule which is atherapeutic agent or a detectable marker. In one embodiment, thetherapeutic agent is a cytotoxic agent, such as a radioisotope. Examplesof radioisotopes for polypeptides include, but are not limited to, ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, and ¹²⁵I, ¹³¹I, ¹⁸⁶Re, and ²²⁵Ac. Methods forpreparing radiolabeled amino acids and related peptide derivatives areknown in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581, U.S. Pat.No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S.Pat. No. RE35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902.For example, a radioisotope may be conjugated by a chloramine T method.In further embodiments, a detectable marker may be a radiolabel, anenzyme, a chromophore, or a fluorescent label.

In a further aspect, the invention relates to an expression vectorencoding a polypeptide, e.g. an antibody, antigen-binding protein orreceptor-Fc fusion protein of the invention. Such expression vectors maybe used for recombinant production of polypeptides of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, anantibody-encoding nucleic acid molecule is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), or a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119. Such nucleic acid vectors and theusage thereof are well known in the art (see, for instance, U.S. Pat.No. 5,589,466 and U.S. Pat. No. 5,973,972).

In another embodiment, the vector comprises the nucleic acid moleculeencoding an antibody or polypeptide of the invention, including anexpression vector comprising the nucleic acid molecules describedwherein the nucleic acid molecule is operatively linked to an expressioncontrol sequence.

In one embodiment, the vector is suitable for expression of apolypeptide or antibody of the invention in a bacterial cell. Examplesof such vectors include expression vectors such as BlueScript(Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264,5503-5509 (1989), pET vectors (Novagen, Madison, Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as yeastalpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al.,ed. Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516-544 (1987)).

A vector comprising a nucleic acid molecule of the invention isprovided, wherein the nucleic acid molecule is operatively linked to anexpression control sequence suitable for expression in a mammalian hostcell.

Expression control sequences are engineered to control and drive thetranscription of genes of interest, and subsequent expression ofproteins in various cell systems. Plasmids combine an expressible geneof interest with expression control sequences (i.e. expressioncassettes) that comprise desirable elements such as, for example,promoters, enhancers, selectable markers, operators, etc. In anexpression vector of the invention, antibody-encoding nucleic acidmolecules may comprise or be associated with any suitable promoter,enhancer, selectable marker, operator, repressor protein, polyAtermination sequences and other expression-facilitating elements.

“Promoter” as used herein indicates a DNA sequence sufficient to directtranscription of a DNA sequence to which it is operably linked, i.e.,linked in such a way as to permit transcription of the antibody-encodingnucleotide sequence when the appropriate signals are present. Theexpression of a antibody-encoding nucleotide sequence may be placedunder control of any promoter or enhancer element known in the art.Examples of such elements include strong expression promoters (e.g.,human CMV IE promoter/enhancer or CMV major IE (CMV-MIE) promoter, aswell as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitinC (UbC), and HIV LTR promoters).

In some embodiments, the vector comprises a promoter selected from thegroup consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi,UbC and HIV LTR.

Nucleic acid molecules of the invention may also be operatively linkedto an effective poly (A) termination sequence, an origin of replicationfor plasmid product in E. coli, an antibiotic resistance gene asselectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise a regulatable induciblepromoter (inducible, repressable, developmentally regulated) as opposedto a constitutive promoter such as CMV IE (the skilled artisan willrecognize that such terms are actually descriptors of a degree of geneexpression under certain conditions).

Selectable markers are elements well-known in the art. Under theselective conditions, only cells that express the appropriate selectablemarker can survive. Commonly, selectable marker genes express proteins,usually enzymes, that confer resistance to various antibiotics in cellculture. In other selective conditions, cells that express a fluorescentprotein marker are made visible, and are thus selectable. Embodimentsinclude beta-lactamase (bla) (beta-lactam antibiotic resistance orampicillin resistance gene or ampR), bls (blasticidin resistance acetyltransferase gene), bsd (blasticidin-S deaminase resistance gene), bsr(blasticidin-S resistance gene), Sh ble (Zeocin® resistance gene),hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM(tetracycline resistance gene or tetR), neomycin phosphotransferase II(npt) (neomycin resistance gene or neoR), kanR (kanamycin resistancegene), and pac (puromycin resistance gene).

In certain embodiments, the vector comprises one or more selectablemarker genes selected from the group consisting of bla, bls, BSD, bsr,Sh ble, hpt, tetR, tetM, npt, kanR and pac. In other embodiments, thevector comprises one or more selectable marker genes encoding greenfluorescent protein (GFP), enhanced green fluorescent protein (eGFP),cyano fluorescent protein (CFP), enhanced cyano fluorescent protein(eCFP), or yellow fluorescent protein (YFP).

For the purposes of this invention, gene expression in eukaryotic cellsmay be tightly regulated using a strong promoter that is controlled byan operator that is in turn regulated by a regulatory fusion protein(RFP). The RFP consists essentially of a transcription blocking domain,and a ligand-binding domain that regulates its activity. Examples ofsuch expression systems are described in US20090162901A1, which isherein incorporated by reference in its entirety.

As used herein “operator” indicates a DNA sequence that is introduced inor near a gene in such a way that the gene may be regulated by thebinding of the RFP to the operator and, as a result, prevents or allowtranscription of the gene of interest, i.e. a nucleotide encoding apolypeptide of the invention. A number of operators in prokaryotic cellsand bacteriophage have been well characterized (Neidhardt, ed.Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol2 ASM Press, Washington D.C. 1996). These include, but are not limitedto, the operator region of the LexA gene of E. coli, which binds theLexA peptide, and the lactose and tryptophan operators, which bind therepressor proteins encoded by the Lad and trpR genes of E. coli. Thesealso include the bacteriophage operators from the lambda P_(R) and thephage P22 ant/mnt genes which bind the repressor proteins encoded bylambda cl and P22 arc. In some embodiments, when the transcriptionblocking domain of the RFP is a restriction enzyme, such as NotI, theoperator is the recognition sequence for that enzyme. One skilled in theart will recognize that the operator must be located adjacent to, or 3′to the promoter such that it is capable of controlling transcription bythe promoter. For example, U.S. Pat. No. 5,972,650, which isincorporated by reference herein, specifies that tetO sequences bewithin a specific distance from the TATA box. In specific embodiments,the operator is preferably placed immediately downstream of thepromoter. In other embodiments, the operator is placed within 10 basepairs of the promoter.

In certain embodiments, the operator is selected from the groupconsisting of tet operator (tetO), NotI recognition sequence, LexAoperator, lactose operator, tryptophan operator and Arc operator (AO).In some embodiments, the repressor protein is selected from the groupconsisting of TetR, LexA, LacI, TrpR, Arc, LambdaC1 and GAL4. In otherembodiments, the transcription blocking domain is derived from aeukaryotic repressor protein, e.g. a repressor domain derived from GAL4.

In an exemplary cell expression system, cells are engineered to expressthe tetracycline repressor protein (TetR) and a protein of interest isplaced under transcriptional control of a promoter whose activity isregulated by TetR. Two tandem TetR operators (tetO) are placedimmediately downstream of a CMV-MIE promoter/enhancer in the vector.Transcription of the gene encoding the protein of interest directed bythe CMV-MIE promoter in such vector may be blocked by TetR in theabsence of tetracycline or some other suitable inducer (e.g.doxycycline). In the presence of an inducer, TetR protein is incapableof binding tetO, hence transcription then translation (expression) ofthe protein of interest occurs. (See, e.g., U.S. Pat. No. 7,435,553,which is herein incorporated by reference in its entirety.)

Another exemplary cell expression system includes regulatory fusionproteins such as TetR-ER_(LBD)T2 fusion protein, in which thetranscription blocking domain of the fusion protein is TetR and theligand-binding domain is the estrogen receptor ligand-binding domain(ER_(LBD)) with T2 mutations (ER_(LBD)T2; Feil et al. (1997) Biochem.Biophys. Res. Commun. 237:752-757). When tetO sequences were placeddownstream and proximal to the strong CMV-MIE promoter, transcription ofthe nucleotide sequence of interest from the CMV-MIE/tetO promoter wasblocked in the presence of tamoxifen and unblocked by removal oftamoxifen. In another example, use of the fusion proteinArc2-ER_(LBD)T2, a fusion protein consisting of a single chain dimerconsisting of two Arc proteins connected by a 15 amino acid linker andthe ER_(LBD)T2 (supra), involves an Arc operator (AO), more specificallytwo tandem arc operators immediately downstream of the CMV-MIEpromoter/enhancer. Cell lines may be regulated by Arc2-ER_(LBD)T2,wherein cells expressing the protein of interest are driven by aCMV-MIE/ArcO2 promoter and are inducible with the removal of tamoxifen.(See, e.g., US 20090162901A1, which is herein incorporated byreference.)

In some embodiments, a vector of the invention comprises a CMV-MIE/TetOor CMV-MIE/A02 hybrid promoter.

The vectors of the invention may also employ Cre-lox tools forrecombination technology in order to facilitate the replication of agene of interest. A Cre-lox strategy requires at least twocomponents: 1) Cre recombinase, an enzyme that catalyzes recombinationbetween two loxP sites; and 2) loxP sites (e.g. a specific 34-base pairby sequence consisting of an 8-bp core sequence, where recombinationtakes place, and two flanking 13-bp inverted repeats) or mutant loxsites. (See, e.g. Araki et al. PNAS 92:160-4 (1995); Nagy, A. et al.Genesis 26:99-109 (2000); Araki et al. Nuc Acids Res 30(19):e103 (2002);and US20100291626A1, all of which are herein incorporated by reference).In another recombination strategy, yeast-derived FLP recombinase may beutilized with the consensus sequence FRT (see also, e.g. Dymecki, S.PNAS 93(12): 6191-6196).

In another aspect, a gene (i.e. a nucleotide sequence encoding arecombinant polypeptide of the invention) is inserted within anexpression-enhancing sequence of the expression cassette, and isoptionally operably linked to a promoter, wherein the promoter-linkedgene is flanked 5′ by a first recombinase recognition site and 3′ by asecond recombinase recognition site. Such recombinase recognition sitesallow Cre-mediated recombination in the host cell of the expressionsystem. In some instances, a second promoter-linked gene is downstream(3′) of the first gene and is flanked 3′ by the second recombinaserecognition site. In still other instances, a second promoter-linkedgene is flanked 5′ by the second recombinase site, and flanked 3′ by athird recombinase recognition site. In some embodiments, the recombinaserecognition sites are selected from a loxP site, a lox511 site, alox2272 site, and a FRT site. In other embodiments, the recombinaserecognition sites are different. In a further embodiment, the host cellcomprises a gene capable of expressing a Cre recombinase.

In one embodiment, the vector comprises a first gene encoding a lightchain of an antibody or a heavy chain of an antibody of the invention,and a second gene encoding a light chain of an antibody or a heavy chainof an antibody of the invention.

In some embodiments, the vector further comprises anX-box-binding-protein 1 (mXBP1) gene capable of enhancing proteinproduction/protein secretion through control of the expression of genesinvolved in protein folding in the endoplasmic reticulum (ER). (See,e.g. Ron D, and Walter P. Nat Rev Mol Cell Bio/0.8:519-529 (2007)).

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g. S. cerevisiae, S.pombe, P. partoris, P. methanolica, etc.), plant cells, insect cells(e.g. SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In certain embodiments, the cell is ahuman, monkey, ape, hamster, rat or mouse cell. In other embodiments,the cell is eukaryotic and is selected form the following cells: CHO(e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g. COS-7), retinal cells,Vero, CV1, kidney (e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21),HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431(epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT cell,tumor cell, and a cell line derived from an aforementioned cell. In someembodiments, the cell comprises one or more viral genes, e.g. a retinalcell that expresses a viral gene (e.g. a PER.C6® cell).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody of the invention as defined herein or a bispecificmolecule of the invention as defined herein. Examples of host cellsinclude yeast, bacterial, and mammalian cells, such as CHO or HEK cells.For example, in one embodiment, the present invention provides a cellcomprising a nucleic acid stably integrated into the cellular genomethat comprises a sequence coding for expression of an antibodycomprising a recombinant polypeptide of the present invention. Inanother embodiment, the present invention provides a cell comprising anon-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid,phagemid, or linear expression element, which comprises a sequencecoding for expression of an antibody comprising the recombinantpolypeptide of the invention. In other embodiments, the presentinvention provides a cell line produced by stably transfecting a hostcell with a plasmid comprising an expression vector of the invention.

In a further aspect, the invention relates to a method for producing anantibody, or antigen-binding protein, or receptor-Fc fusion protein ofthe invention, said method comprising the steps of a) culturing ahybridoma or a host cell of the invention as described herein above, andb) purifying the antibody, or antigen-binding protein, or receptor-Fcfusion (supra) from the culture media.

In an even further aspect, the invention relates to a compositioncomprising: an antibody or antigen-binding fragment thereof,antigen-binding protein or receptor-Fc fusion protein as defined herein,or a bispecific molecule as defined herein.

The compositions may be formulated with pharmaceutically acceptablecarriers or diluents as well as any other known adjuvants and excipientsin accordance with conventional techniques such as those disclosed inRemington: The Science and Practice of Pharmacy, 19th Edition, Gennaro,Ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosenantibody of the present invention and the chosen mode of administration.The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve theappropriate stability of drug substance, desired therapeutic responsefor a particular patient, composition, and mode of administration. Theselected dosage level will depend upon a variety of pharmacokineticfactors.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering an antibody of the presentinvention in vivo are well known in the art and may be selected by thoseof ordinary skill in the art. (Daugherty, A L, and Msrny, R J, Adv DrugDelivery Rev, 58(5-6): 686-706 (2006)).

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases or disorders. The inventionprovides for the detection or diagnosis of a disease or disorder,comprising: (a) assaying the existence of antigen in cells or tissuesamples of a subject using one or more antibodies thatimmunospecifically bind to the target antigen; and (b) comparing thelevel of the antigen with a control level, e.g. levels in normal tissuesamples, whereby an increase in the assayed level of antigen compared tothe control level of antigen is indicative of the disease or disorder,or indicative of the severity of the disease or disorder.

Antibodies of the invention can be used to assay antigen levels in abiological sample using immunohistochemical methods well-known in theart. Other antibody-based methods useful for detecting protein includeimmunoassays such as the enzyme linked immunoassay (ELISA) and theradioimmunoassay (RIA). Suitable antibody labels may be used in suchkits and methods, and labels known in the art include enzyme labels,such as alkaline phophatase and glucose oxidase; radioisotope labels,such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹²¹In), and technetium (^(99m)Tc); and luminescent labels, suchas luminol and luciferase; and fluorescent labels, such as fluoresceinand rhodamine.

Presence of labeled antibodies may be detected in vivo for diagnosispurposes. In one embodiment, diagnosis comprises: a) administering to asubject an effective amount of a labeled antibody; b) waiting for a timeinterval following administration for permitting labeled antibody toconcentrate at sites where antigen may be detected and to allow forunbound labeled antibody to be cleared to background level; c)determining a background level; and d) detecting the labeled antibody inthe subject, such that detection of labeled antibody above thebackground level is indicative that the subject has the disease ordisorder, or is indicative of the severity of the disease or disorder.In accordance with such embodiment, the antibody is labeled with animaging moiety suitable for detection using a particular imaging systemknown to those skilled in the art. Background levels may be determinedby various methods known in the art, including comparing the amount oflabeled antibody detected to a standard value previously determined fora particular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

EXAMPLES

The following examples are provided to describe to those of ordinaryskill in the art how to make and use methods and compositions of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure the accuracywith respect to numbers used (e.g. amounts, concentrations, temperature,etc.) but some experimental errors and deviations should be accountedfor.

Example 1 Preparation of the Recombinant Polypeptides

Generating the recombinant polypeptides, for example a chimeric C_(H)IgG4 (SEQ ID NO: 31) and a chimeric C_(H) IgG1 (SEQ ID NO: 30), was doneusing standard cloning techniques. First, the chimeric IgG4 C_(H) wasgenerated through a two-step PCR amplification process. Two PCRfragments, Fragment 1 and 2, were amplified using starting constructpR85501 (containing a wild-type hIgG4 C_(H) DNA) and using primersoSP030-oSP031 and oSP032-oSP033 (see Table 8), respectively. The primersintroduced the desired IgG2 lower hinge sequence (which encodes SEQ IDNO:3) and the flanking restriction sites into the fragments. These twofragments were then joined using PCR primers oSP031 and oSP033. Theresulting sequence was inserted into pR85501via XhoI-NotI restrictionsites generating a vector construct pR85502 that contains a chimericIgG4 C_(H) having an IgG2 lower hinge sequence. The sequence wasconfirmed using primers KO_oLRC120 and oKO021.

In addition, a chimeric IgG1 C_(H) was generated through multiple stepPCR amplification. Fragment 1a was generated using primers oSP031 andoSP035 (see Table 8 below) from template pR85503 (which contains awild-type human IgG1 C_(H) DNA). Fragment 2a was amplified with primersoSP036 and oSP038 using pR85502 (containing the chimeric IgG4 C_(H) DNA)as a template. Fragment 3 was made using primers oSP037 and oSP039 fromtemplate pR85503 (wild-type hIgG1 C_(H) DNA). Fragments 1a and 2a werejoined using primers oSP031 and oSP038, which generated Fragment 4.Joining Fragments 2a and 3 using primers oSP036 and oSP039 createdFragment 5. Fragment 4 and 5 were then fused using primers oSP031 andoSP039. The resulting sequence was inserted into pR85501 via XhoI-NotIrestriction sites generating a construct pR85504 that has an IgG1constant region with the IgG2 lower hinge and IgG4 CH2 domain. Thesequence was confirmed using primers KO_oLRC120 and oKO021.

TABLE 8Primers for PCR generation of chimeric C_(H )nucleic acid constructsPrimer name Primer Sequence (SEQ ID NO) oSP0305′-TTCGCGCAGCTTAGGTTTATGCCAGGGGGGACGGGTGGCACGGGTCGTGGTGGACACCGT- 3′(antisense) (SEQ ID NO: 16) oSP0315′-AAGCTTATACTCGAGCTCTAGATTGGGAACCCGGGTCTCT-3′ (SEQ ID NO: 17) oSP0325′-CCCACCGTGCCCAGCACCACCTGTGGCAGGACCATCAGTCTTCCTGTTCCCCCCAAAA-3′(SEQ ID NO: 18) oSP0335′-TGTGTCTTCAGGGAGAGGGACAGAGACCCATTTACTCGCC GGCG-3′ (antisense)(SEQ ID NO: 19) oSP0355′-CTCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGTGGACACCGT- 3′(antisense) (SEQ ID NO: 20) oSP0365′-AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCACCTGTG-3′(SEQ ID NO: 21) oSP0375′-GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC-3′(SEQ ID NO: 22) oSP0385′-CTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTG GTGTCCACATGTGG-3′(antisense) (SEQ ID NO: 23) oSP0395′-CTTCAGGGAGAGGGACAGAGGCCCATTTACTCGCCGGCG-3′ (antisense)(SEQ ID NO: 24) KO_oLRC120 5′-GCTGACAGACTAACAGACTG-3′ (SEQ ID NO: 25)KO_Fc-4-3 5′-GACCTCAGGGGTCCGGGAGATCAT-3′ (SEQ ID NO: 26) oKO0215′-ATACATTATACGAAGTTATACCGGTA-3′ (SEQ ID NO: 27) oKO0145′-GTGAGCGCTCTTCGGCAGACGTCCAACTGGTGCAGTCAGGG-3′ (SEQ ID NO: 39) oKO0155′-CAGCTAGCTCTTCCGGCTGAGGAGACGGTGACCGTGGTGCCTTGGCC-3′ (SEQ ID NO: 40)

Example 2 Generation of Chimeric Heavy Chain Antibodies

Exemplary antibodies were obtained using standard methodologies. Ananti-hCD3 antibody (anti-hCD3 antibody “L2K”) was used to construct thechimeric antibodies of this example. L2K was obtained by well-knownmethods based on WO2004/106380. Anti-hCD3_L2K, designated herein asControl Antibody 1, contains a wild-type human IgG1 heavy chain constantregion (SEQ ID NO:13).

The bispecific antibodies created in accordance with the present Examplecomprise two separate antigen-binding domains (i.e., binding arms). Ananti-hCD3×anti-hCD20 (Bispecific antibody), designated herein as ControlAntibody 2, was obtained as described in US Patent ApplicationPublication No. US20100331527A1. The bispecific antibody was constructedusing standard methodologies wherein a heavy chain and a light chainfrom an “L2K” anti-CD3 antibody of WO2004/106380 were combined with aheavy chain from an anti-CD20 antibody (see e.g. PCT InternationalApplication No. PCT/US13/60511, filed on Sep. 19, 2013, which is hereinincorporated by reference in its entirety). Control Antibody 2 containswild-type human IgG4 heavy chain constant regions (SEQ ID NO:15), yetthe anti-CD3 arm has a modified CH3 domain (SEQ ID NO:42) for ease ofpurification.

Control Antibody 3 was obtained using the same methodologies asdescribed herein to combine the variable regions of Control Ab 2(Anti-hCD3×anti-hCD20 Bispecific Ab) with a wild-type human IgG1 heavychain constant regions (SEQ ID NO:13), having a modified CH3 domain (SEQID NO:41) in the anti-CD3 heavy chain arm.

Control Antibody 4 contains an antigen-binding domain capable of bindingCA9 antigen and a wild-type human IgG1 heavy chain constant region (SEQID NO:13).

Control Antibody 5 is an anti-hCD3×anti-hCD20 bispecific antibodyobtained according to the methods of PCT International Application No.PCT/US13/60511, filed on Sep. 19, 2013, which is herein incorporated byreference in its entirety. Briefly, a first antigen-binding domaincomprising a heavy chain variable region derived from an anti-CD20antibody (“CD20-VH”) is paired with a light chain variable regionderived from an anti-CD3 antibody (“CD3-VL”). The CD20-VH/CD3-VL pairingcreates an antigen-binding domain that specifically recognizes CD20. Asecond antigen-binding domain comprising a heavy chain variable regionderived from an anti-CD3 antibody (“CD3-VH”) is paired with a lightchain variable region derived from an anti-CD3 antibody (“CD3-VL”). TheCD3-VH/CD3-VL pairing creates an antigen-binding domain thatspecifically recognizes CD3. Control Antibody 5 is engineered withwild-type human IgG1 heavy chain constant regions (SEQ ID NO:13), yetthe anti-CD3 arm has a modified CH3 domain (SEQ ID NO:41) in the heavychain constant region for ease of purification.

Isotype controls were made that do not bind to the same target antigenas the tested antibodies, e.g. do not bind CD3 or CD20 antigen.Wild-type IgG1 Isotype Control contains the wt heavy chain constantregion amino acid sequence of SEQ ID NO:13. Wild-type IgG4 “CPPC”Isotype Control has the wt CH amino acid sequence of SEQ ID NO:15,except having the “CPPC” hinge mutation S228P (according to EUnumbering).

The constant region of Control Antibody 1 was replaced with a chimerichuman constant region of the invention, e.g SEQ ID NO: 31 or SEQ ID NO:30. Replacement of the constant region was done by obtaining a L2Kvariable region nucleic acid sequence (plasmid pR85505) that wasamplified using primers oKO014 and oKO015 (see Table 8). The L2Kvariable region (SEQ ID NO: 34) was then introduced into plasmid pR85502using Sap1 restriction site for cloning. The sequence of the resultingplasmid pR85506 was confirmed using primers KO_oLRC120 andoKO_Fc_(—)4-3. This construct was used to generate Antibody 1 of theinvention, sIgG4-anti-CD3_L2K (also known herein as sIgG4) (whichcomprises SEQ ID NO:31), using standard methodologies for isolatingantibodies.

In a second example, the L2K variable region was amplified using primersoKO014 and oKO015 (see Table 8) using plasmid pR85505 as template. Thevariable region was then introduced into plasmid pR85504 using Sap1restriction site for cloning. The sequence of the resulting plasmidpR85507 was confirmed using primers KO_oLRC120 and oKO_Fc_(—)4-3. Thisconstruct was used to generate Antibody 2 of the invention,sIgG1-anti-CD3_L2K (also known herein as sIgG1) (which comprises SEQ IDNO:30), using standard methodologies.

Antibody 3 was constructed from the anti-CD3×anti-CD20 bispecificantibody of Control Antibody (Ab)₅. Control Ab 5 had its heavy chainconstant regions replaced with chimeric constant heavy chain regions,the anti-CD20 arm having an heavy chain constant region amino acidsequence comprising SEQ ID NO: 30, and the anti-CD3 arm having amutation in the CH3 domain of the CH (SEQ ID NO:37) to create Antibody 3(also known herein as sIgG1*).

Similarly, Antibody 4 was created from bispecific antibody Control Ab 5whereas heavy chain constant regions were replaced with chimeric CH, theanti-CD20 arm having an heavy chain constant region amino acid sequencecomprising SEQ ID NO: 31, and the anti-CD3 arm having a mutation in theCH3 domain of the CH (SEQ ID NO:38) to create Antibody 4 (also knownherein as sIgG4*).

The chimeric antibodies comprising constant regions of SEQ ID NO:30 orSEQ ID NO:31 (or bispecific antibodies comprising SEQ ID NO:30/37 or SEQID NO:31/38), and the control antibodies, were used in certainexperiments set out in the Examples that follow.

Example 3 Chimeric Antibodies Specifically Bind to Jurkat Cells

After chimeric antibodies were converted to fully human IgGs, specificantigen binding properties were determined. The example antibodyconstructs and control antibodies, as set forth in Example 2, weretested using fluorescence-activated cell sorting (FACS) for theirability to bind to Jurkat cells (human T-cell line expressing targetantigens CD3+, CD20). FACS data was acquired using the followingprotocol: Cells at 2×10⁵ per well were incubated with serially dilutedantibodies and 100 μl supplements for 1 hour at 4° C. Post incubation,cells were washed twice and appropriate secondary antibodies (e.g.fluorescent-tagged FITC anti-human IgG) were added and incubated for anadditional 30 minutes at 4° C., then washed twice. Cells werere-suspended in cold PBS containing 1% BSA and analyzed by flowcytometry on a FACSCanto II™ flow cytometer (BD Biosciences). Jurkatcells were gated by side and forward scatter sorting. Each EC₅₀ for cellbinding titration was determined using Prism (GraphPad Software, SanDiego, Calif.) with values calculated using a 4-parameter non-linearregression analysis.

It was determined that chimeric antibodies bind to Jurkat cells at equalconcentrations compared to control antibodies having a wild-type C_(H)region, therefore chimeric antibodies with altered C_(H) regions havenot lost their ability to bind antigen. See FIG. 6.

Example 4 Characterization of Antibodies-Binding to U937 Cells

U937 cells, a monocyte cell line expressing FcγRI and FcγRIIA, wereplated and allowed to incubate with serial dilutions of Ab (the highestconcentration of Ab used is 50 nM). Cells were incubated with Abs for 1hr at 4° C. then washed twice. U937 cells were then incubated withsecondary Ab (FITC goat anti-human Fab) for 30 min at 4° C. then washedtwice. Cells were analyzed by flow cytometry using standard methods andmedian fluorescent intensity (MFI) was recorded. Results are summarizedin Table 9 and FIG. 7 where it is demonstrated that chimeric Abs,Antibody 1 (sIgG4) and Antibody 2 (sIgG1), bind to U937 cells at highconcentrations.

TABLE 9 Binding of Chimeric Abs vs. Wild-type Abs to U937 cells Antibody(Ab) EC₅₀ (nM) Control Ab 1 1.3 sIgG1 (Ab2) 45.4 Control Ab 2 0.91 sIgG4(Ab 1) 33.5

Example 5 Characterization of Antibodies-U937 cytotoxic assay

U937 cells were used as a positive killer effector control in thefollowing cytoassay. As such, the ability of antibodies with chimericC_(H) regions to kill U937 cells via Fc/FcγR interactions was tested.Calcein killing assays were carried out using the following protocol:Human and cynomolgus Peripheral Blood Mononuclear Cells (PBMCs) wereisolated over Ficoll-Paque (GE Healthcare Life Sciences) or viaLympholyte-Mammal density cell separation media (CedarlaneLaboratories), respectively. The isolated PBMCs were activated over acourse of several days with media containing recombinant human IL-2 (30U/ml) and T-cell activation beads (anti-CD3/CD28 for human PBMC,anti-CD2/CD3/CD28 for cynomolgus PBMC). Activated T-cells were isolatedfrom the PBMCs by centrifugation, then resuspended in 1 ml media. Themagnetized beads were removed from the T-cells. Target cells (U937) werelabeled with calcein, then washed and followed by incubation with theisolated activated T-cells (10:1 effector:target ratio) and antibody,using 3-fold serial dilutions of antibody over a course of 3 hours at37° C. Following incubation, the plates were centrifuged andsupernatants were transferred to a translucent black clear bottom platefor fluorescence analysis. Each EC₅₀, defined as the molar concentrationof antibody that induces 50% cytotoxicity, was determined using Prism(GraphPad Software, San Diego, Calif.). Values were calculated using a4-parameter non-linear regression analysis. Results are summarized inFIG. 8.

The cytotoxic activity of Antibody 1 (sIgG4) and Antibody 2 (sIgG1) issignificantly diminished as compared to corresponding antibodiescontaining wild-type IgG4 and IgG1 hinge regions. See FIG. 8.Interestingly, although the chimeric Abs weakly bind at higherconcentrations as shown in Example 4, they do not kill U937 cells in thecytoassay.

Example 6 Characterization of Antibodies-Proliferation of hPBMCs

The ability of chimeric antibodies and control constructs to stimulatePeripheral Blood Mononuclear Cells (PBMCs) and induce proliferation wasassessed using ATP catalyzed quantification (CellTiter GIGO). Theactivation of PBMCs results in the release of cytokines, which drivecellular proliferation. Proliferation data was acquired using thefollowing protocol: Human or cynomolgus monkey derived PBMC (5×10⁵/well)were incubated with a 3-fold serial dilution of anti-CD20×CD3 or Controlantibody (including Control Ab 4 specific to CA9 antigen) in 96 wellplates for 72 h at 37° C. Following incubation, CellTiter Glo® was addedand luminescence was measured using a VICTOR X5 multi-label plate reader(PerkinElmer). The EC₅₀ of cell viability (ATP catalyzed quantification)was determined using Prism (GraphPad Software, San Diego, Calif.).Values were calculated using a 4-parameter non-linear regressionanalysis and are shown in FIG. 9.

Antibody 1 (sIgG4) and Antibody 2 (sIgG1) do not activate cellularproliferation in comparison to corresponding antibodies containingwild-type IgG4 and IgG1 hinge regions. See FIG. 9.

Example 7 Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Chimeric Antibodies

The anti-CD3×anti-CD20 bispecific antibodies having chimeric constantheavy chain regions sIgG1* (Antibody 3) and sIgG4* (Antibody 4) wereanalyzed using Surface Plasmon Resonance (SPR) (Biacore) technology todetermine their kinetic binding parameters to human and cynomolgus Fcγreceptors. Isotype controls, namely wt-IgG1 Isotype Control and wt-IgG4CPPC Isotype Control, were tested in a similar manner.

Briefly, SPR experiments were performed at 25° C. on a Biacore T200instrument employing a carboxymethyl dextran-coated (CM-5) chip. A mousemonoclonal anti-penta-histidine antibody (GE Healthcare) was immobilizedon the surface of the CM-5 sensor chip using standard amine-couplingchemistry. 140RU-376RU of His-tagged human or monkey FcγR proteins werecaptured on the anti-penta-histidine amine-coupled CM-5 chip and stocksolutions of antibodies were injected at 20 μl/min for 2.5 min over thecaptured proteins. mAb binding response was monitored and, for lowaffinity receptors, steady-state binding equilibrium was calculated.Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t_(1/2) (min)=(ln 2/(60*k_(d)).

TABLE 10 Kinetic binding parameters for wild-type (wt) and chimericheavy chain antibodies Binding to His-captured human FcγRI at 25° C.Antibody k_(a) (M⁻¹sec⁻¹) k_(d) (¹sec⁻¹) K_(D) (10⁻⁹M) T^(1/2) (min)wt-IgG1 1.74E+05 7.48E−04 4.3 15 Isotype Control wt-IgG4 CPPC 1.71E+052.36E−03 13.9 5 Isotype Control sIgG1* (Ab 3) NB NB NB NB sIgG4* (Ab 4)NB NB NB NB NB: No binding

As the results in Table 10 demonstrate, sIgG1* and sIgG4* bispecificantibodies display no binding to human FcγRI, compared to antibodieshaving the wild-type (wt) hIgG1 or hIgG4-CPPC CH region. Chimeric heavychain antibodies of this invention also display weak to no binding forseveral of the low-affinity FcRγ receptors (e.g. FcRγIIa, FcRγIIb)compared to antibodies with wt hIgG1 or hIgG4-CPPC Fc sequence (Table11).

TABLE 11 Steady-state equilibrium binding for wild-type (wt) andchimeric heavy chain antibodies Binding to His-captured low-affinityhuman and cynomolgus FcγR receptors at 25° C. K_(D) (10⁻⁶M) Values forLow Affinity FcyR Binding to Chimeric Heavy Chain Antibodies human humanhuman human Antibody hFcyRIIA FcyRIIA cynomolgus human cynomolgusFcyRIIIA FcyRIIIA cynomolgus human Tested (H131) (R131) FcyRIIA FcyRIIBFcyRIIB (V176) (F176) FcyRIIIA FcyRIIIB wtIgG1 1.1 2 4.2 2 4.2 1.5 1.30.6 2.3 Isotype Control wtIgG4 12 10 19.3 9.8 9.6 10 26 5.8 NB (CPPC)Isotype Control sIgG1* 11.7 20.5 23.5 233 14.6 NB NB 42.4 NB (Ab 3)sIgG4* 12 19.3 23.1 123 13.9 NB NB 66.3 NB (Ab 4) NB: No binding

Example 8 IgG1 and IgG4 Antibodies Having Chimeric CH Regions ShowDecreased Effector Function in CDC Assay

Antibodies with chimeric CH regions (sIgG1* and sIgG4*), as describedabove, were generated to produce mAbs with altered or reduced effectorfunction. Compared to antibodies comprising a wild-type (wt) heavy chainconstant region of the IgG1 isotype, amino acid substitutions in the CHregion may hinder the ability of an Ig Fc to bind to its receptor.Hence, signaling and immune responses, such as B cell activation orphagocytosis, may be altered. The effect of amino acid modifications inthe CH region on complement dependent cytoxicity (CDC) (in this example)and antibody-dependent cytoxicity (ADCC) effector function (see Example9) was examined.

To examine the effect of Antibody 3 (sIgG1*) and Antibody 4 (sIgG4*) onCDC effector function, CD20-expressing Raji (target) cells (5000/well)or Daudi cells were plated in the presence of 5% human serum complement.Serial dilutions of sIgG1*, sIgG4* and control antibodies, starting at100 nM, were added to cells for 4 h at 37° C. Target cell lysis wasdetermined using the CytoTox Glo™ kit (Promega) and percent cytoxicitywas calculated.

Percent cytotoxicity was calculated using the equation: %cytotoxicity=((L_(S)−L_(SR))/(L_(MR)−L_(SR)))*100% where L_(sR) isbaseline target cell luminescence and L_(MR) is maximal calcein releasefrom cells lysed by digitonin. The EC₅₀ for cytoxicity was determinedusing Prism software (GraphPad). Values were calculated using4-parameter non-linear regression analysis and are shown in Table 12,and FIGS. 10A and 10B.

The CDC activity of Antibody 3 (sIgG1*) and Antibody 4 (sIgG4*) againstDaudi and Raji cells is significantly diminished as compared tocorresponding antibody having a wt heavy chain constant domain. SeeTable 12, and FIGS. 10A/B. Some CDC activity was observed with sIgG4*against Raji cells, however, overall results show that the chimericantibodies mount weaker effector responses than wt IgG1 Fc controlantibodies.

TABLE 12 sIgG1* and sIgG4* antibodies display reduced activity in CDCassays measuring effector function CDC Target Cell: Raji Daudi MaximumMaximum Cytotoxicity EC50 [M] Cytotoxicity (%) EC50 [M] (%) Control6.12E−08 ~95 1.98E−08 ~85 Ab 5 Ab 3 NA NA 3.49E−08 ~10 (sIgG1*) Ab 4 NANA 2.86E−08 ~45 (sIgG4*) NA: No activity

Example 9 IgG1 and IgG4 Antibodies Having Chimeric CH Regions ShowDecreased Effector Function in ADCC Assay

To examine the effect of Antibody 3 (sIgG1*) and Antibody 4 (sIgG4*) vs.antibodies with wild-type CH regions on ADCC effector function, freshlyisolated unstimulated CD56-positive NK cells or NK92 cells engineered toexpress the higher affinity V allele of FcγRIIIa were plated withCalcein-labeled CD20-positive Raji or Daudi cells in the presence ofchimeric CH-antibodies and wt-CH control antibodies. Calcein releasefrom target cells was monitored and percent cytoxicity was determined.Percent cytotoxicity and EC₅₀ were calculated as described for CDCassay, above. Results are shown in Table 13 and FIGS. 11A and 11B.

The chimeric CH antibodies, sIgG1* and sIgG4*, do not mediate ADCCactivity (Table 13) against Raji or Daudi cells.

TABLE 13 sIgG1* and sIgG4* antibodies display reduced activity in ADCCassays measuring effector function ADCC Target Cell: Raji Daudi MaximumMaximum Cytotoxicity EC50 [M] Cytotoxicity (%) EC50 [M] (%) Control Ab 51.87E−10 ~70^(#) 1.48E−09 ~65^(#) Ab 3 (sIgG1*) NA NA NA NA Ab 4(sIgG4*) NA NA NA NA NA: No activity; ^(#)background cytotoxicity ~20%

Example 10 Pharmacokinetic Profile of Chimeric Antibodies

The toxicokinetic profile of Antibody 3 (also known herein as sIgG1*)and Antibody 4 (also known herein as sIgG4*) was evaluated by obtainingblood samples from male cynomolgus monkeys (3 animals/treatment group)receiving a single 30-minute IV infusion, followed by a 12-weekobservation period. Blood samples for toxicokinetic analysis of totaldrug concentrations in serum were collected pre-dose and post-dose at 5minutes, and 5, 24, 48, 72 and 168 hours, and Day 14, 21, 35, 49, 66 and84. The resultant serum samples were analyzed by a direct enzyme linkedimmunosorbent assay (ELISA) to determine the total drug concentration ofthe sIgG1* or sIgG4* antibody. The toxicokinetics of the test articleswere assessed using non-compartmental analysis (Phoenix WinNonLin) todetermine pharmacokinetic parameters. Results are shown in Table 14(AUC=area under the concentration vs. time curve; C_(max)=observedmaximum concentration in serum).

TABLE 14 Pharmacokinetic Profile of Chimeric Antibodies in Serum ofCynomolgus monkeys Following a Single Intravenous Infusion to CynomolgusMonkeys 1 mg/kg 1 mg/kg sIgG1* sIgG4* Parameter Units Mean SD Mean SDC_(max) μg/mL 33.4 3.79 26.0 4.72 C_(max)/Dose kg * μg/mL/mg 33.4 3.7926.0 4.72 t_(max) day 0.0243 0 0.0243 0 AUC_(0-168 h) day · μg/mL 10020.1 61.1 8.04 AUC_(0-168 h)/ day * kg * ug/mL/ 100 20.1 61.1 8.04 Dosemg T½ Day 8.14 1.15 14.0 2.64

Following a single intravenous dose of 1.0 mg/kg of sIgG1* and sIgG4* incynomolgus monkeys, mean peak concentrations (C_(max)) of 33.4 and 26.0μg/mL, respectively, and mean AUC_(0-168h) values of 100 and 61.1day*ug/mL, respectively, were observed. The apparent terminal half-lifewas estimated to be between 8.14-14.0 days of these two molecules. Thedata indicate that continuous exposure to sIgG1* and sIgG4* wasmaintained in all animals for the majority of the 12-week observationperiod and exposure was comparable across treatment groups. No apparentimmunogenicity with the test articles was observed. The overallpharmacokinetic profiles of sIgG1* and sIgG4* are typical of monoclonalantibodies dosed in cynomolgus monkey.

What is claimed:
 1. A recombinant polypeptide comprising a heavy chainconstant (CH) region comprising, from N-terminus to C-terminus, a CH1domain, a chimeric hinge, a CH2 domain, and a CH3 domain wherein: (a)the CH1 domain comprises the amino acid sequence DKKV or DKRV frompositions 212 to 215 (EU numbering), (b) the chimeric hinge comprises ahuman IgG1 or a human IgG4 upper hinge amino acid sequence frompositions 216 to 227 (EU numbering) and a human IgG2 lower hinge aminoacid sequence PCPAPPVA (SEQ ID NO: 3) from positions 228 to 236 (EUnumbering), (c) the CH2 domain comprises a human IgG4 CH2 domain aminoacid sequence from positions 237 to 340 (EU numbering), and (d) the CH3domain comprises a human IgG1 or a human IgG4 CH3 domain sequence frompositions 341 to 447 (EU numbering).
 2. The recombinant polypeptide ofclaim 1, wherein the CH region has an amino acid sequence at least 99%identical to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 37, or SEQ ID NO:
 38. 3. The recombinantpolypeptide of claim 1, wherein the CH1 domain comprises the amino acidsequence DKKV (SEQ ID NO: 4), and the chimeric hinge comprises the aminoacid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8).
 4. The recombinantpolypeptide of claim 1, wherein the CH1 domain comprises the amino acidsequence DKRV (SEQ ID NO: 5), and the chimeric hinge comprises the aminoacid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9).
 5. The recombinantpolypeptide of claim 1, wherein the CH2 domain comprises the amino acidsequence SEQ ID NO:
 10. 6. The recombinant polypeptide of claim 1,wherein the CH3 domain comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 11, or SEQ ID NO: 12, SEQ ID NO: 41,and SEQ ID NO:
 42. 7. The recombinant polypeptide of claim 1, whereinthe recombinant polypeptide is an antigen-binding protein.
 8. Therecombinant polypeptide of claim 1, wherein the recombinant polypeptideis a receptor-Fc fusion protein.
 9. The recombinant polypeptide of claim1, wherein the recombinant polypeptide is an antibody.
 10. Therecombinant polypeptide of claim 9, wherein the antibody exhibits directcytotoxic activity of less than 20% cytolysis, or less than 10%, or 5%,4%, 3%, 2%, or even 0% or undetectable cytolysis, at an antibodyconcentration of at least 10 nM.
 11. The recombinant polypeptide ofclaim 10, wherein the direct cytotoxic activity is at least about 5-foldless, or at least about 10-fold less than the direct cytotoxic activityof a corresponding antibody comprising a wild-type IgG1 or wild-typeIgG4 CH region.
 12. A recombinant polypeptide, wherein the polypeptidecomprises N′-VD1-X1-Y1-Y2-X2-X3-C′, wherein: (a) N′ is the N-terminusand C′ is the C-terminus of the polypeptide, (b) VD1 is an amino acidsequence comprising an antigen-binding domain, (c) X1 is an amino acidsequence comprising a domain selected from the group consisting of anIgG1 CH1 domain, an IgG4 CH1 domain, and at least positions 212-215 (EUnumbering) of an IgG1 or IgG4 CH1 domain, (d) Y1 comprises an amino acidsequence from positions 216-227 (EU numbering) of an IgG1 or IgG4 hingeregion, (e) Y2 comprises the human IgG2 lower hinge region amino acidsequence PCPAPPVA (SEQ ID NO: 3) from positions 228 to 236 (EUnumbering), (f) X2 is an amino acid sequence comprising an IgG4 CH2domain, and (g) X3 is an amino acid sequence comprising an IgG1 CH3domain or an IgG4 CH3 domain.
 13. The recombinant polypeptide of claim12, wherein the polypeptide comprises an amino acid sequence at least99% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 37, or SEQ ID NO:
 38. 14. The recombinant polypeptide ofclaim 12, wherein X1 comprises the amino acid sequence DKKV (SEQ ID NO:4), and Y1-Y2 comprises a chimeric hinge consisting of the amino acidsequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 8).
 15. The recombinantpolypeptide of claim 12, wherein X1 comprises the amino acid sequenceDKRV (SEQ ID NO: 5), and Y1-Y2 comprises a chimeric hinge consisting ofthe amino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 9).
 16. Therecombinant polypeptide of claim 12, wherein X2 comprises the amino acidsequence of SEQ ID NO:
 10. 17. The recombinant polypeptide of claim 12,wherein X3 comprises the amino acid sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 41, and SEQ IDNO:
 42. 18. The recombinant polypeptide of claim 12, wherein therecombinant polypeptide is an isolated antigen-binding protein.
 19. Therecombinant polypeptide of claim 12, wherein the recombinant polypeptideis a receptor-Fc fusion protein.
 20. The recombinant polypeptide ofclaim 12, wherein the recombinant polypeptide is an isolated antibody.21. The recombinant polypeptide of claim 20, wherein the antibodyexhibits direct cytotoxic activity of less than 20% cytolysis, or lessthan 10%, or 5%, 4%, 3%, 2%, 2%, or even 0% or undetectable cytolysis,at an antibody concentration of at least 10 nM.
 22. The recombinantpolypeptide of claim 21, wherein the direct cytotoxic activity is atleast about 5-fold less, or at least about 10-fold less than the directcytotoxic activity of a corresponding antibody comprising a wild-typeIgG1 or wild-type IgG4 CH region.
 23. A composition comprising arecombinant polypeptide of claim 1 or claim
 12. 24. A nucleic acidmolecule encoding a recombinant polypeptide of claim 1 or claim
 12. 25.The nucleic acid molecule of claim 24, wherein the recombinantpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, and SEQ ID NO:31, SEQ ID NO: 37, and SEQ ID NO:
 38. 26. The nucleic acid molecule ofclaim 24 having a nucleotide sequence greater than 99% sequence identityto the nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO:
 36. 27. The nucleic acidmolecule of claim 25 comprising a nucleotide sequence selected the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ IDNO: 33, SEQ ID NO: 35, and SEQ ID NO:
 36. 28. A vector comprising anucleic acid molecule claim
 25. 29. The vector of claim 28, wherein thenucleic acid molecule is operatively linked to an expression controlsequence suitable for expression in a host cell.
 30. The vector of claim29, wherein the expression control sequence comprises a promoterselected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, UbC,RSV, SL3-3, MMTV, Ubi and HIV LTR.
 31. The vector of claim 29, whereinthe expression control sequence comprises an expression cassetteselected from the group consisting of TetR-ER_(LBD)T2 fusion gene drivenby the CMV-MIE promoter, blasticidin resistance gene driven by the SV40promoter, and Arc-ER_(LBD)T2 fusion gene driven by the CMV-MIE promoter.32. The vector of claim 30, wherein the promoter is a CMV-MIE/TetO orCMV-MIE/Arc hybrid promoter.
 33. The vector of claim 30, comprising oneor more selectable marker genes selected from the group consisting ofbla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR and pac.
 34. Acell comprising a nucleic acid of claim
 24. 35. A cell comprising thevector of claim
 28. 36. The cell of claim 34, wherein the nucleic acidis integrated into the genome of the cell.
 37. The cell of claim 36comprising a nucleic acid encoding a protein expression enhancer. 38.The cell of claim 37 comprising a nucleic acid encoding an XBPpolypeptide.
 39. The cell of claim 34, wherein the cell is a eukaryoticcell.
 40. The cell of claim 34, wherein the cell is an animal cell. 41.The cell of claim 34, wherein the cell is a mammalian cell.
 42. The cellclaim 34, wherein the cell is a CHO cell.
 43. The cell of claim 34,wherein the cell is a CHO-K1 cell.